Starch

ABSTRACT

This invention relates to a method of producing a starch with unique functionality in plants through mutagenesis, and/or using biotechnology, and/or breeding practices. Further the invention relates to the starch from maize plants and/or other plants which produce starch storing organs which contain low amylose starch which has an amylose content between 1.5% and 15% and preferably between 1.5% and 10% and most preferably 1.5 and 8%. The invention includes starch extracted from such novel grain due to at least one mutation induced by ethyl methanesulfonate. Additionally, the invention uses a biotechnology approach involving controlling the activity of the granule bound starch synthase enzyme in starch storing organ. The invention includes the use of the starch for its cooking, paste, and gel properties.

This application is a divisional of allowed U.S. patent application Ser.No. 10/272,291, filed Oct. 17, 2002, which is based on, and claims thebenefit of, U.S. Provisional Application No. 60/329,525, filed Oct. 17,2002, the entire contents of which are incorporated herein by reference

The present invention relates to a starch defined herein as elasticstarch. The presently disclosed starch has been made possible byengineering the waxy locus of starch producing plants, or the geneproduct of the waxy locus (i.e., the GBSS protein), which synthesizesamylose. The starch of the present invention therefore may be viewed asa reduced amylose starch or a special type of waxy starch with newelastic properties. The starch of the present invention is referred toherein as waxy-E or wx-E starch, to emphasize this elastic property notpreviously available with known waxy starch. In particular, the starchof the present invention has special properties of high viscosity andvaluable paste and gel properties not previously found in natural i.e.,wild type) starches or waxy starches of plants of similar species. Thespecial properties of the starches of the present invention are believedto be the product of the unique combination of reduced amylose contentof the starch of the present invention, as compared to starch of a wildtype plant of the same species, and a similar amylopectin structure ofthe starch of the present invention, as compared to starch of a wildtype plant of the same species. While these special properties have beencharacterized herein using a Rapid Fisco Analyzer, one of ordinary skillin the art will appreciate that other means are available tocharacterize the physical properties which may be used to describe thepresently disclosed starch. The starch of the present invention may beobtained from plants and/or plant parts through mutagenesis or by planttransformation or other approaches known in the art to reduce theamylose content of plants without affecting amylopectin structure andwithout reducing amylose content as significantly as is found in waxystarches which have little or no amylose. Further, the invention relatesto a method of increasing the elasticity of a formulation by utilizing awaxy-E starch of the present invention.

Chemically, starch can be described as a mixture of two homoglucosepolymers: amylose and amylopectin. Amylose is a generally linear α-1,4glucan which sometimes is lightly-branched with α-1,6-glycosidiclinkages. Amylopectin is normally larger than amylose and ishighly-branched with α-1,6-glycosidic linkages. The balance of amyloseand amylopectin in normal starches isolated from storage tissues likepotato tubers or cereal grain is normally between 20 to 30 percentamylose and the remainder 70 to 80 percent is described as amylopectinon a dry starch weight basis.

Plants displaying altered starch storing organ phenotypes have beenimportant in advancing our understanding of how starch is produced inplants. For example, numerous phenotypes have been reported for amize(Glover and Mertz, 1987, Corn, in Agronomy. American Society ofAgronomy, Madison; Coe et al, 1988, The genetic of corn, in Corn andCorn Improvement, 3^(rd) edition, G. F. Sprague and J. W. Dudley, eds.American Society of Agronomy, Madison) and several phenotypes (e.g.,waxy, amylose extender, dull, shrunken, sugary-2, and sugary) have beendescribed extensively with regard to their effects on carbohydratecomposition and response to genetic background, allelic dosage, orinteraction with other mutations (example references: Creech, 1965,Genetics 52:1175-1186; Holder et al, 1974, Crop Science 14:643-646;Garwood and Vanderslice, 1982, Crop Science 22:367-371; Garwood et al,1976, Cereal Chemistry 53:355-364). Many studies of starch storing organphenotypes have focused on the molecular structure of synthesizedpolysaccharides and the concentration and type of soluble carbohydratesfound in the starch storing organ during early-to-mid development. Inparticular, examination of maize starch storing organs with differingphenotypes have been instrumental in characterizing carbohydratemetabolism in cereal grain and determining which enzymes have a role inregulating starch biosynthesis (for review see Boyer, 1985,Phytochemistry 24:15-18; Shannon and Garwood, 1984, Starch: chemistryand Technology; R. L. Whistler, J. N. BeMiller, and E. F. Paschall, eds;Academic Press, Orlando).

Across all plants, one starch storing organ phenotype produces a starchwhich contains a low quantity of amylose. This phenotype is called“waxy” starch for historical reasons: in maize the phenotype of theintact seed has a waxy phenotypic appearance. Plants producing waxystarch are often referred to as waxy plants or waxy mutants; the gene iscommonly referred to as the waxy gene. Granule bound starch synthase[GBSS-ADPglucose: 1,4-α-D-glucan-4-α-D-glucosyltransferase(E.C.2.4.1.21)] enzyme activity is strongly correlated with the productof the waxy gene (Shure et al, 1983, Cell 35: 225-233). The synthesis ofamylose in a number of species such as maize, rice and potato has beenshown to depend on the expression of this gene (Tsai, 1974, BiochemicalGenetics 11: 83-96; Hovenkamp-Hermelink et al, 1987, Theoretical andApplied Genetics 75: 217-221). Visser et al described the molecularcloning and partial characterization of the gene for granule-boundstarch synthase from potato (1989, Journal of Plant Science 64:185-192).Visser et al (1991, Molecular and General Genetics 225289-296) have alsodescribed the inhibition of the expression of the gene for GBSS inpotato by antisense constructs. Further, starch synthases (EC 2.4.1.11and EC 2.4.1.21) elongate starch molecules (Delrue et al, 1992,Bacteriology 174:3612-3620; Denyer et al, 1999a, Biochemical Journal340:183-191; Denyer et al, 1999b, Biochemical Journal 342:647-653) andare thought to act on both amylose and amylopectin. Starch synthase[SS-ADP glucose: 1,4-α-D-glucan-4-α-D-glucosyltransferase (EC2.4.1.11)]activity can be found associated both with the granule an in the stromaof the plastid. The capacity for starch association of the bound starchsynthase enzyme is well known. Various enzymes involved in starchbiosynthesis are now known to have differing propensities for binding asdescribed by Mu-Forster et al (1996, Plant Physiology 111:821-829). Theother SS enzymes have become known as soluble starch synthases,following the pioneering work of Frydman and Cardini (Frydman andCardini, 1964, Biochemical and Biophysical Research Communications17:407-411). Recently, the appropriateness of the term “soluble” hasbecome questionable in light of discoveries that these enzymes areassociated with the granule as well as being present in the solublephase (Denyer et al, 1993, Plant Journal 4:191-198; Denyer et al., 1995,Planta 97:57-62; Mu-Forster et al., 1996, Plant Physiology 111:821-829).It is generally believed that the biosynthesis of amylopectin involvesthe interaction of soluble starch synthases and starch branchingenzymes. Different isoforms of soluble starch synthase have beenidentified and cloned in pea (Denyer and Smith, 1992, Planta186:609-617; Dry et al, 1992, Plant Journal, 2:193-202), potato (Edwardset al, 1995, Plant Physiology 112:89-97; Marshall et al, 1996, PlantCell 8:1121-1135) and in rice (Baba et al, 1993, Plant Physiology103:565-573), while barley appears to contain multiple isoforms, some ofwhich are associated with starch branching enzyme (Tyynela and Schulman,1994, Physiologica Plantarum 89:835-841). In maize, two soluble forms ofSS, known as isoforms I and II, have been identified (Macdonald andPreiss, 1983, Plant Physiology 73:175-178; Boyer and Preiss, 1978,Carbohydrate Research 61:321-334; Pollock and Preiss, 1980, Archives ofBiochemistry and Biophysics 204:578-588; Macdonald and Preiss, 1985Plant Physiology 78: 849-852; Dang and Boyer, 1988, Phytochemistry27:1255-1259; Mu et al, 1994, Plant Journal 6:151-159), but neither ofthese has been cloned. SSI activity of maize endosperm was found to becorrelated with a 76-kDa polypeptide found in both soluble andgranule-associated fractions (Mu et al., 1994, Plant Journal 6:151-159).The polypeptide identity of SSII remains unknown.

Waxy maize starch which contains essentially no amylose has been knownfor many years (Shannon and Garwood, 1984; Starch: Chemistry andTechnology; R. L. Whistler, J. N. BeMiller, and E. F. Paschall, eds;Academic Press, Orlando; pp 50-56). There are examples of such waxystarches in peas, maize, rice, potato, sorghum, wheat, barley and otherplants.

For many plants including wheat, pleas, corn, and potatoes among others,a principal purpose for their domestication and cultivation is forstarch production. The utilization of the starch may be in the form ofthe intact starch storing organ itself (e.g., a baked potato) or as apreparation of a substantially complete starch storing organ (e.g. flouror meal or sliced potatoes). Alternatively, the starch may be isolatedfrom starch storing organs for incorporation into foodstuffs (e.g. piefillings, puddings, soups, sauces, gravies, coatings, candies and/orconfectionary products, and/or yoghurts and other dairy products) and/orindustrially-derived products (e.g. paper sizing aids, textile sizingaids, and/or suspension aids). Starch is produced in plants as granules:microscopic structures with spherical, elliptical, or polyhedral shapeswhich contain individual starch molecules.

Examination of the color that starch stains with the addition of iodineis one of the simplest methods of identifying waxy starches. Whenstained with iodine, normal starch will stain blue or purple. A waxystarch will be red or brown or brownish-red in color when stained withiodine because the amylose component is severely reduced such that thereis little, or essentially no amylose present. Waxy starches have beenconsistently described as (a) nearly 100% amylopectin or (b) isolatedfrom plant starch storage organs which lack a GBSS enzyme in theendosperm or (c) from plant starch storage organs which have originatedfrom a plant which produces a starch which is nearly 100% amylopectin or(d) from plant starch storage organs which have originated from a plantwhich lacks a GBSS enzyme in the endosperm or (e) having some or all ofthese qualities or (f) having unknown or undocumented quality (U.S. Pat.Nos. 4,428,972; 4,615,888; 4,767,849; 4,789,557; 4,789,738; 4,801,470;6,143,963).

Some waxy starches might stain blue or purple and may appear to containsome amylose as a result of changes in amylopectin structure. Forexample, in maize long-chain amylopectin is produced due to the decreasein starch branching enzyme activity as a result of the amylose-extendermutation in the starch biosynthetic pathway (Boyer et al, 1976, Journalof Heredity 67:209-214). Waxy amylose-extender starch, a starch which isproduced in plants having both waxy and amylose-extender mutations, mayhave an apparent amylose content of 15% to 26% (Shannon and Garwood,1984; Starch: Chemistry and Technology; R. L. Whistler, J. N. BeMiller,and E. F. Paschall, eds; Academic Press, Orlando; p 65). The differencesin the structure of waxy starch and waxy amylose-extender starch, andthe effects of the amylose-extender mutation on starch in general, areclearly observed in the distribution of their component chains (Jane etal, 1999, Cereal chemistry 76:629-637). This, and other alterations ofthe starch biosynthetic pathway, have an effect on amylopectin starchstructure and starch cooking, gelling, pasting, and in general, starchTheological properties.

Thus, starch granules, which have a blue coloration, contain longchains. The long chains may either be real amylose or a component of theamylopectin of the starch as a result of an alteration in the starchbiosynthetic pathway (e.g. the amylose-extender mutation in maize),resulting in an apparent amylose content by some methods and no amyloseby others (Klucinec and Thompson, 1998, Cereal chemistry 75:887-896).Additionally, amylopectin and waxy starch may appear to have an amylosecontent of 5% itself by quantitative iodine staining methods. Thisamylose may be attributed to the low iodine binding capacity of theamylopectin and may be falsely attributed to amylose when the iodinebinding capacity of the amylopectin is not taken into considerationduring measurements (Knutson and Grove, 1994, Cereal Chemistry71:469-471).

Much time and effort has been spent to produce waxy starch, which stainsred by virtue of the fact that in this form it has very little amylose.Waxy starch and normal starch differ in the way they change during acooking process. Heating starch in water or an aqueous solution resultsin changes in the starch granules (Whistler and Daniel, 1985,Carbohydrates, in Food Chemistry, O. R. Fennema, ed., Marcel Dekker,Inc., New York, pp. 114-115). During heating, granules swell and theorganized structures maintaining the granule structure dissociate,permitting further swelling. With additional heating and applied shearforces, granules will eventually collapse to form an unorganized pasteof starch molecules. This process of starch granule swelling anddissociation, known as gelatinization, is known to those familiar withthe art (Atwell et al, 1988, Cereal Foods World 33(3):306-311; Testerand Morrison, 1990, Cereal Chemistry 67:551-557). Upon cooling, starchbegins to reorganize into structures resembling those which originallyheld the starch granules together, however the complete highly organizedstructure of the granule is never reestablished. This process ofreorganization, known as retrogradation, is well known to those familiarwith the art (Atwell et al, 1988, Cereal Foods World 33(3):306-311).Retrogradation often involves changes in the physical properties of thestarch paste, including a decrease in paste clarity and gelation of thepaste. Normal starches are generally recognized for their ability to gelwithin hours (Ring, 1985, Starch/Stärke 37; 80-83), while waxy starchesare generally recognized for their ability to require weeks to gel ifthey gel at all (Yuan and Thompson, 1998, Cereal Chemistry 75:117-123;Biliaderis, 1992, Characterization of starch networks by small straindynamic oscillatory rheometry, in Developments in CarbohydrateChemistry, R. J. Alexander an H. F. Zobel, eds., American Association ofCereal Chemists, St. Paul, p 103). Normal starches are generallyrecognized for forming opaque pastes and gels, while waxy starches aregenerally recognized for remaining transparent after processing (Craiget al, 1989, Cereal Chemistry 66:173-182). Waxy starches are considereduseful as water binders, viscosity builders, and texturizers in food aswell as industrial applications (Reddy and Seib, 2000, Journal of CerealScience 31:25-39). Waxy starches also have better freeze-thaw stabilityand clarity compared to normal starches once cooked (Whistler andBeMiller, 1997, Carbohydrate chemistry for Food Scientists, Eagan Press,St. Paul, p. 146; Reddy and Seib, 2000, Journal of Cereal Science31:25-39). Waxy starches are also less resistant to shear, acid, andhigh temperatures than are normal starches, and extended cooking of waxystarches results in stringy, cohesive pastes (Whistler and BeMiller,1997, Carbohydrate chemistry for Food Scientists, Egan Press, St. Paul,p. 142; Reddy and Seib, 2000, Journal of Cereal Science 31:25-39). Thesecharacteristics of waxy starch are believed to be a result of themolecular characteristics of the starch, specifically the absence ofamylose (Whistler and Daniel, 1985, Carbohydrates, in Food Chemistry, O.R. Fennema, ed., Marcel Dekker, Inc., New York, p. 113), though theprecise behavior of the starch also depends on the concentration of thestarch and the conditions under which it is processed and subsequentlystored. Finally, it is generally recognized that it is common for waxystarch to be chemically modified by substitution, crosslinking, or both,to improve its stability to temperature, shear and acid as well as tominimize its undesirable paste qualities (Whistler and Daniel, 1985,Carbohydrates, in Food Chemistry, O. R. Fennema, ed., Marcel Dekker,Inc., New York, pp. 118-120). Such practices are common to thosefamiliar with the art (Zheng, G. H. et al, 1999, Cereal Chemistry76:182-188; Reddy ad Seib, 2000, Journal of Cereal Science 31:25-39).

By eliminating other key starch biosynthesis enzymes, other alterationsof the starch biosynthetic pathway can result in useful starches.Several patents exist on the creation and use of such starches (U.S.Pat. Nos. 4,428,972; 4,615,888 4,767,849; 4,789,557; 4,789,738;4,801,470; 5,009,911; and 5,482,560). More recently, several patents andpublished applications have described the production and utilization ofheterozygous combinations of mutations in the starch biosyntheticpathway to obtain commercially useful starches (WO95/35026, U.S. Pat.Nos. 5,356,655; 5,502,270; and 5,516,939). The production of many ofthese starches involves the use of double or triple mutant plants. Inthese cases in which waxy starch is involved the inventors have statedthat “plants homozygous recessive for the waxy gene lack a granule boundstarch synthase [GBSS] enzyme and produce nearly 100% amylopectin” (U.S.Pat. Nos. 5,356,655; 5,502,270). Due to the number of mutations requiredto sufficiently alter the starch (at least 2 or 3 within a single plant)many of these starches are difficult and costly to produce commercially,so many of these starches from plants with mutations in the starchbiosynthetic pathway are uncompetitive with chemically modifiedstarches. Further, these combinations of 2 or more mutations, whetherthey are combined homozygously or heterozygously in the plant endosperm,rely on the alteration of the structure of amylopectin from normal orwaxy starch.

Waxy potato starches have been shown to contain an amylose content aslow as 0% and as high as 7.9% (Salehuzzaman et al, 1999, Plant, Cell,and Environment 22:1311-1318, van der Leij et al, 1991, Theoretical andApplied Genetics 82:289-295). However, the amylose content of all ofthese starches is regarded as zero (van der Leij et al, 1991,Theoretical and Applied Genetics 82:289-295). Hovenkamp-Kermelink et al(1987, Theoretical and Applied Genetics 75:217-221) produced a waxymutant of potato by screening microtubers produced from plants exposedto X-ray radiation. The starch from two microtubers was found to have anamylose content of approximately 5%, but a second generation of tubersproduced from additional microtubers from the same irradiated plantsresulted in starch with a normal amylose content. Examination of anadditional set of tubers resulted in three tubers, two of which staineda solid reddish-brown characteristic of the waxy mutation Neuffler etal, 1997, Mutants of Maize, Cold Spring Harbor Laboratory Press,Plainview, N.Y., p. 298) and a third which stained a mixture of reddishbrown and blue indicating a heterogeneous mixture of waxy starch andamylose-containing starch of unknown quality within the potato tuber.The waxy potatoes did not produce a GBSS enzyme. No distinction was madebetween these starches with an amylose content below 3.5%. Van der Leijet al (1991, Theoretical and Applied Genetics 75:217-221) observed thatpotato starches could have an amylose content of between 3% and 7.9% andthe tubers would stain red with iodine stain, a primary characteristicof waxy starches. No distinctions were made between these starcheshaving an amylose content between 3% and 7.9%.

Studies have produced antisense transgenic potatoes having amylosecontents between 3.0% and 8% (van der Leij et al, 1991, Theoretical andApplied Genetics 82:289-295; Visser et al, 1991, Molecular and GeneralGenetics 225:289-296; Kuipers et al, 1994, Plant Cell 6:43-52) infurther attempts to understand the function and activity of GBSS. Theamylose contents of these starches were shown to be a result of tuberswith both blue and red-brown staining portions (Visser et al, 1991,Molecular and General Genetics 225:289-296), indicating heterogeneousmixtures of waxy starch and amylose-containing starch of unknownquality. Kuipers et al (1994, Plant Cell 6:43-52) also observedheterogeneity on a granule level, with starch granules having blue coresand surrounded by a red-brown colored shell of starch, with the size ofthe blue core increasing in size with an increase in the amylose contentof the starch. Further, the elastic properties and gelling abilities ofpastes, and the gel properties of gels produced from these low amylosestarches are unknown. Studies have attempted to restore the productionof amylose in waxy potato plants by transforming the plants with genesfor GBSS enzymes produced by other plants. Salehuzzman et al (1999,Plant Cell and Environment 22:1311-1318) partially restored amylose toamylose free mutants of potato to between 3.5% and 13% amylose bytransformation with the cassaya GBSS enzyme with different amyloplasttransit peptides. For starches between 3.5% and 13% amylose, thestarches produced were heterogeneous mixtures of amylose-containingstarch and red-brown staining waxy starch: the starch granules had bluecores surrounded by a red-brown colored shell of starch, with the sizeof the blue core increasing in size with increases in the amylosecontent of the starch (Salehuzzman et al, 1999, Plant Cell andEnvironment 22:1311-1318). Salehuzzaman et al (1999, Plant Cell andEnvironment 22:1311-1318) additionally observed that a paste of a potatostarch with an apparent amylose content of 13% developed an elasticmodulus during cooling while a paste of a waxy potato starch did not;the elastic behavior of the heterogeneous starches with lower amylosecontents were not reported. Waxy potatoes transformed with GBSS isoformsfrom pea resulted in potatoes with amylose contents of between 0.8% an1%, and like the other low amylose potatoes and pea starch,heterogeneity was observed within the granules: granules stained withiodine stain revealed amylose in concentric rings or havingblue-staining granule cores (Edwards et al, 2002, The Plant Cell14:1767-1785). The presence of the amylose produced by pea GBSS wasclaimed to have an effect on the cooking properties of the starch(Edwards et al., 2002, The Plant Cell 14:1767-1785), however thedifferences observed between the starches are within the errorassociated with this type of instrumental measurement. Flipse et al(1996, Theoretical and Applied Genetics 92:121-127) extracted starchfrom plants produced from crosses between a waxy potato and a normalpotato; the potato tubers had varying levels of GBSS activity and nolinear correlation was observed between GBSS activity and amylosecontent. Starches with amylose contents of 2.50%, 16.94%, 18.96%, and20.32% were examined for their swelling properties and the rheologicalproperties of swollen starch granules. No clear differences of theeffect of amylose were observed in the swelling and rheologicalproperties of the granules. The only conclusion that could be made wasthat the presence of amylose (above 16.94%) had an influence on thephysical behavior of the granules.

Thus, in potato, reduction in the amylose content of the starch hasresulted in the production of heterogeneous mixtures ofamylose-containing starch and waxy starch, with heterogeneity among apopulation of starch granules and within individual starch granules.Further, no distinctions in the physical properties of waxy starcheswith amylose contents between 0% and 7.9% have been made. Thus, from theexisting literature it may be inferred that for potato starch, amylosecontents of less than 7.9% confer no unique Theological or pastingproperties to these starches outside of those properties observed foreither waxy potato or normal potato starch. Further, the elasticproperties and gelling abilities of pastes, and the gel properties ofgels produced from starches below 13% amylose are unknown, and thosetests which have been conducted indicate that the physical propertiesare within the error associated the physical properties of waxy potatostarch or a potato starch with a normal amylose content.

Like the transgenic potato starches, pea mutants producing starch withamylose contents lower than normal pea starch produced granule with bluecores and a red-brown periphery (Denyer et al, 1995, Plant Cell andEnvironment 18:1019-1026), indicating that they were heterogeneousmixtures of amylose-containing starch and waxy starch. Cooking, paste,and gel behavior was not reported for these starches.

Extensive work initially in Japan has identified waxy wheat starches.The range of amylose content of these waxy mutants was narrow, beingapproximately 0.5% difference between the highest level and the lowestlevel reported. In all cases the starch was reported as staining redwith iodine and the amylose content was reported as zero or near zeropercent. A waxy wheat starch was also created using mutagenesis of adouble-null wheat known as “Ike” to generate a non-null wheat(WO09815621) which stained red when tested with iodine stain. A nullallele does not produce a certain protein at that allele on a certainchromosome, and a null mutant does not produce a certain protein at anyof the chromosomes. This is in contrast to a non-null mutant which doesproduce the protein. Further work with transgenic lines has found thatdisruption of the waxy gene using antisense technology can produce lineslacking in amylose. In all cases these lines were screened for iodinecoloration and red-brown staining starches were found and selected outof the transformants.

Miura and Sugawara (1996, Theoretical and Applied Genetics 93:1066-1070)have shown that substitution of genes producing functional GBSS enzymewith the null alleles can result in starches with a 22 to 23% amylosecontent rather than the 25.5% amylose content of the normal control.Likewise, Miura et al (1999, Euphytica 108:91-95) have shown thatelimination of the functionality in two of the three GBSS enzymeisoforms in wheat endosperm results in a wheat starch which has anamylose content of at least 16% and more often between 20% and 21% ofthe normal 25% amylose present in the starch. Thus, the presence of onewild type GBSS enzyme is sufficient to produce a starch with an amylosecontent of at least 16%. Oda et al (1992, Japanese Journal of Breeding42:151-154) has shown that low amylose wheat starches having an amylosecontent between 14.1 and 16.7% can be created through ethylmethanesulphonate (EMS) mutagenesis of the seeds. Sasaki et al (2000,Cereal Chemistry 77:58-63) produced wheat starches with amylose contentsof about 7.5% and 13.5% by crossing normal wheat with waxy wheat. Peakviscosities of all starches differed by less than 20% of the peakviscosity of the waxy wheat starch, with the low amylose starches havinga higher peak viscosity than both normal and waxy wheat starch. Thegelatinization temperatures and enthalpy were highest for waxy wheatsand decreased in the order waxy>13.5% amylose wheat>7.5% amylosewheat>normal wheat starch. The retrogradation temperatures and enthalpywere insignificantly different for waxy wheat, normal wheat, or any ofthe low amylose wheat starches. From retrogradation data, the inferencethat these low amylose wheat starches exhibit unique Theologicalproperties could not be made. Further, the elastic properties andgelling abilities of pastes, and the gel properties of gels producedfrom any of these low amylose starches are unknown. Additionally, inthis case since the low amylose trait is not fixed in one wheat line,but instead is the product of two lines with widely differing amylosecontents, the resultant low amylose seed if grown will not produce seedswith one type of low amylose starch but instead will produce a mixtureof seeds containing starch having a range amylose contents varyingwidely between those of the original waxy and normal parents. Thesestarches made from crosses of normal plants and waxy plants are not thesubject of the present invention.

Kiribuchi-Otobe et al (1998, Cereal Chemistry 75:671-672) found thatstarch granules extracted from a wheat strain derived from mutagenizedTanikei A6099 had an apparent amylose content of 1.6% and stained darkbrown with dark cores compared to red-staining waxy wheat starch (0.4%apparent amylose). This same wheat was claimed to have an amylosecontent of 0.8% to 2.5% in U.S. Pat. No. 6,165,535 to presumably accountfor the approximately 1% error associated with the amylose contentassay. Kiribuchi-Otobe et al (1998, Cereal Chemistry 75:671-672) foundthat this mutant wheat starch had an initial high temperature viscositystability relative to a waxy wheat starch (0.4% amylose). However, theviscosity of the starch paste decreased dramatically, to the sameviscosity as the waxy wheat, during continued cooking and remained atthe same viscosity as waxy wheat after cooking. The mutagenized TanikeiA6099 wheat is known to produce a mutant GBSS enzyme (Yanagisawa et al,2001, Euphytica 121:209-214), but the effect of the mutation on theactivity of the enzyme is not known (Yanagisawa et al, 2001, Euphytica121:209-214). Additionally, it is unknown whether the starch containstrue amylose, which normally would result in a blue coloration withiodine stain rather than a dark brown stain for this mutant starch, orcontains a modified amylopectin structure. The act of mutagenesis itselfmay have created other mutations in the plant genome which could haveadditional effects on biosynthesis and thus the cooking properties ofthe starch (e.g. the amylose-extender mutation in maize), and thestructure of amylopectin is also clearly known to have a significantimpact on the paste and gel properties of a starch (Jane et al, 1999,Cereal Chemistry 76:629-637). These other enzymes are known to thoseworking in the area of starch biosynthesis, biochemistry, and chemistry.Further, it has been suggested that GBSS may influence the structure ofamylopectin as well (Martin and Smith, 1995, The Plant Cell 7:971-985),and a mutation in GBSS could conceivably result in an enzyme whichpreferentially produces an altered amylopectin rather than synthesizeamylose. Thus, alteration of the amylopectin structure of the starch mayalso affect starch cooking and rheological properties. Kiribuchi-Otobeand colleagues (U.S. Pat. No. 6,165,535; Kiribuchi-Otobe et al, 1998,Cereal Chemistry 75:671-672; Yanagisawa et al, 2001, Euphytia121:209-214) have not shown that their plants produce an active GBSS norhave they shown that their starch contains amylose and/or produces anormal wheat amylopectin. Further, the elastic properties and gellingabilities of pastes, and the gel properties of gels produced from thislow amylose wheat starch are unknown.

Thus, in wheat lines, reduction in the amylose content of the starch hasresulted in the production of heterogeneous mixtures of brown-stainingstarch of unknown amylose and amylopectin quality relative to normalwheat starch. Further, no distinctions in the rheological properties ofstarches with amylose contents between 1.6% and 15% have been made.Thus, from the existing literature the rheological properties ofstarches with amylose contents between 1.6% and 15% from hybrid wheatplants are unknown. Some evidence suggests that wheat starches having7.5% or 13.5% amylose may have some unique cooking properties, butproduction of these starches was a result of hybridization andrecombinations of genetics which cannot be carried uniformly into futuregenerations of material. Further, the elastic properties and gellingabilities of pastes, and the gel properties of gels produced from wheatstarches below 1.6% and 15% amylose are unknown.

Low amylose sorghum starches have been shown to contain up toapproximately 5% apparent amylose, though these low amylose sorghumstarches are commonly referred to as waxy sorghum starches. Horan andHeider (1946, Cereal chemistry 23:492-503) indicated that some waxysorghum starches had an amylose content as high as 5%, however theyadmitted that the method they utilized to determine the amylose contentswas primarily used to differentiate waxy from normal sorghum starch andwas a rapid method subject to large errors. Miller and Burns (1970,Journal of Food Science 35:666-668) also found waxy sorghums to containup to approximately 5% amylose, and no distinction was made between this5% amylose starch and the waxy sorghum starches with amylose contentsbelow 1%. Thus, it may be inferred that for sorghum a small quantity ofamylose apparently confers no special cooking or rheological qualitiesto these starches.

Waxy starches and low amylose rice starches have been shown to containbetween 0% and 3% amylose, though collectively these starches arereferred to as waxy rice starches (Reyes et al, 1965, Journal ofAgricultural and Food Chemistry 13:438-442; Juliano et al, 1969, Journalof Agricultural and Food Chemistry 17:1364-1369; Sanchez et al, 1988,Cereal chemistry 65:240-243). With these waxy rice starches, it has beenassumed that the differing cooking and paste properties of thesestarches are due to differences in the structure of the amylopectin ofthe starch rather than the amylose content of the starch (Wang and Wang,2002, Cereal Chemistry 79:252-256). Thus, it may be inferred from theliterature that for rice, reduced levels of amylose compared to normalstarches confers no special cooking or other rheological qualities tothese starches. The effects of amylose and other molecular andcompositional characteristics of rice starches on rice (Champagne et al,1999, Cereal Chemistry 76:764-771; Bett-Garber et al, 2001, CerealChemistry 78:551-558) or rice starch properties remain unclear (Lai etal, 2000, Cereal Chemistry 77:272-278).

Low amylose rice starches have been shown to have amylose contentsbetween 7% and 15% (Kumar and Khush, 1988, Euphytica 38:261-269).Shimada et al (1993, Theoretical and Applied Genetics 86:665-672)produced several antisense rice plants with starch having amylosecontents between 6% and 13%. The iodine staining qualities of thesestarch granules were not reported. Further, any cooking properties ofthe starches, the elastic properties of gels produced from these lowamylose rice starches produced by transgenic rice plants are unknown.

Sano (1984, Theoretical and Applied Genetics 68:467-473) and Sano et al(1986, Euphytica 35:1-9) investigated the effects of two alleles on thegene expression at the waxy locus in rice. The Wx_(b) allele was shownto relate to ineffective production of GBSS enzyme and amylose, whilethe Wx_(a) allele was shown to produce larger quantities of GBSS enzymeand amylose. Villareal et al (1989, Starch 41:369-371) also showed thatthe Wx^(a) allele was less effective in the production of amylose thanthe Wx^(b) allele based on analysis of 40 rice varieties. Additionally,Isshiki et al (1998, Plant Journal 15:133-138) observed that for twowild-type rice alleles, Wx^(a) and Wx^(b), Wx^(b) had a GBSS activitytenfold lower than Wx^(a) at the protein and mRNA levels. The decreasein the activity of Wx^(b) compared to Wx^(a) was the result of a pointmutation within the genetic sequence for the normal rice enzyme (Wx^(a)allele). The Wx^(b) allele resulted in the synthesis of a 3.4 kilobasepair mRNA transcript compared to a 2.3 kilobase pair mRNA transcript forWx^(a) as a result of the inclusion of an intron into the mRNA sequenceas a result of the point mutation. Starch produced from rice plants wasrelated to the ability of the plant to excise the intron from the mRNAsequence. Plants which expressed high levels of mature mRNA (withoutintron 1) and no pre-mRNA (containing intron 1) produced the highestlevels of GBSS protein and the highest levels of amylose (20.0 to 27.8%amylose). With more balanced expression of mature and pre-mRNA, lowerlevels of GBSS protein and amylose were observed (6.7 to 16.0% amylose).When all of the mRNA contained intron 1, and no mature mRNA wasobserved, no GBSS protein was observed and no amylose was detected (Wanget al, 1995, Plant Journal 7:613-622). This pattern relating amylosecontent to mature mRNA with properly excised intron 1 could be appliedacross 31 different rice cultivars (Wang et al, 1995, Plant Journal7:613-622). Thus based on the work of Shimada et al (1993, Theoreticaland Applied Genetics 86:665-672), Isshiki et al (1998, Plant Journal15:133-138), and Wang et a (1995, Plant Journal 7:6213-622), low amyloserice appears to be the result of a decrease in the amount of normal GBSSthrough a mutation which results in problems with mRNA processing ratherthan due to a mutation in the mature mRNA sequence. Further, no clearrelationships exist between rice and rice starch properties and amylosecontent.

Waxy corn starches are considered to stain red by iodine stain accordingto the Maize Genome Database [supported by the United States Departmentof Agriculture, Agricultural Research Service (USDA-ARS), the NationalScience Foundation (NSF), and the University of Missouri]. Numerousdominant mutant alleles producing an active GBSS protein (Table 1) andrecessive mutant alleles producing waxy starch (Table 2) exist. Inmaize, it is well known that increasing the dosage of the wx mutation inthe endosperm of the seeds decreases the amylose content of the starch,but seeds with two doses of the wx mutation (out of a possible three inthe triploid endosperm) produce a starch with an apparent amylosecontent near 18% I the mature seed compared to 23-25% amylose in thestarch isolated from normal seed (Sprague et al, 1943, Journal of theAmerican Society of Agronomy 35, 817-822; Boyer et al, 1976, Journal ofHeredity 67:209-214). TABLE 1 Dominant mutant alleles of Waxy (Wx)Wx1-m8-r10 Wx1-m8r1 Wx1-m8r2 Wx1-m9-r3 Wx1-m9-r4 Wx1-m9r1 Wx1-Mo17Wx1-Mt42 Wx1-N28(Ht) Wx1-NC258 Wx1-NC268 Wx1-NC296 Wx1-NC298 Wx1-NC300Wx1-NC304 Wx1-Oh07B Wx1-Oh40B Wx1-Oh43 Wx1-Os420 Wx1-P39 Wx1-Pa91Wx1-R177 Wx1-R213 Wx1-R4 Wx1-RobA Wx1-SA24 Wx1-SC213 Wx1-SC76 Wx1-SG1533Wx1-T218 Wx1-T232 Wx1-T8 Wx1-Tx303 Wx1-Tx601 Wx1-U267Y Wx1-Va102Wx1-Va22 Wx1-Va35 Wx1-Va59 Wx1-Va99 Wx1-W117Ht Wx1-W153R Wx1-W182BWx1-W22 Wx1-W22Cs Wx1-W23 Wx1-W64A Wx1-WF9 Wx1-Wf9 Wx1 Wx1-38-11 Wx1-AWx1-A12 Wx1-A188 Wx1-A554 Wx1-A619 Wx1-A632 Wx1-A634 Wx1-A635 Wx1-A641Wx1-B14A Wx1-B164 Wx1-C49A Wx1-B2 (Missouri) Wx1-B52 Wx1-B68 Wx1-B73Wx1-B37 Wx1-B77 Wx1-B84 Wx1-B95 Wx1-B76 Wx1-C103 Wx1-C11 Wx1-C123Wx1-B97 Wx1-CI187-2 Wx1-Ky228 Wx1-CM37 Wx1-CM105 (Canada) Wx1-CO159Wx1-D940Y Wx1-DE811 Wx1-CMV3 Wx1-EP1 Wx1-F2 Wx1-F2834T Wx1-E2558WWx1-GT112 Wx1-GT119 Wx1-H95 Wx1-F44 Wx1-HP301 Wx1-HY Wx1-Hy Wx1-H99Wx1-I205 Wx1-I29 Wx1-IA2132 Wx1-I137TN Wx1-IDS91 Wx1-IL677A Wx1-K55Wx1-IDS28 Wx1-Ki14 Wx1-Ky21 Wx1-Ky226 Wx1-K64 Wx1-L317

TABLE 2 Recessive mutant alleles of waxy (wx) wx1-m7::Ac7wx1-m7::inactive wx1-m8::Spm-l8 wx1-m8311B::Ds wx1- wx1-m86246xwx1-m9::Ac wx1-m9::Ds m844::En1 wx1-m9::Ds-cy wx1-mCS10::Dswx1-mCS13::Ds wx1-mCS14::Ds wx1- wx1-MCS16::Ds wx1-MCS17::Dswx1-mCS18::Ds mCS15::Ds wx1- wx1-MCS20::Ds wx1-mCS22::Ds wx1-mCS23::DsmCS19::Ds wx1- wx1-MCS7::Ds wx1-MCS8::Ds wx1-mCS9::Ds mCS24::Ds wx1-Mo17wx1-Mum1 wx1-Mum10 wx1-Mum11 wx1-Mum2 wx1-Mum3 wx1-Mum4 wx1-Mum5::Muwx1-Mum6 wx1-Mum7 wx1-Mum8 wx1-Mum9 wx1-Mus16 wx1-Mus181 wx1-Mus215wx1-N1050A wx1-N1240A wx1-P60 wx1-R wx1-S15 wx1-S5 wx1-S9 wx1-Stonor wx1wx1-11 wx1-12 wx1-21 wx1-84-4 wx1-90 wx1-a wx1-Alexander wx1-B wx1-B1wx1-F wx1-B3-S1 wx1-B2::TouristA wx1-B3r wx1-B4::Ds2 wx1-B5 wx1-B6wx1-B7 wx1-B73 wx1-B8 wx1-BL2 wx1-BL3 wx1-C wx1-c wx1-C1 wx1-C2 wx1-C3wx1-C31 wx1-C34 wx1-C4 wx1-CY wx1-B3::Ac wx1- Ds6(U66842) wx1-G wx1-Hwx1-H21 wx1-I wx1-J wx1-M wx1-L wx1- K::Hopscotch wx1-m1::Ds wx1-m32::Bgwx1-m6R wx1- m5:8313delta14 wx1-m6-o1 wx1-m6::Ds wx1-m6NRwx1-m5:8313::Ds

In the early 1940's, a waxy mutant (wx^(a)) was discovered in two exoticArgentinean small-seeded flint corn varieties which contained a starchwhich ha an amylose content of 2.4% (Brimhall et al, 1945, Journal ofthe American Society of Agronomy 37:937-944). The starch stained a paleviolet color (Brimhall et al, 1945, Journal of the American Society ofAgronomy 37:937-944). Additionally, the amylose content of the starchincreased from 0% (wax) to 0.65% to 1.3% to 2.4% (full wx^(a)) withincreasing dose of the trait when the plant bearing the starch wascrossed with a waxy plant (Brimhall et al, 1945, Journal of the AmericanSociety of Agronomy, 37:937-944; Sprague and Jenkins, 1948, Iowa StateCollege Journal of Science 22:205-213), Echt and Schwartz (1981, MaizeGenetics Cooperation Newsletter 55:8-9) described the wx-a allele asresulting in a 95% reduction in the amount of GBSS protein produced anda starch with a low amylose content. Examination of cooked starch showedthat the viscosity of the paste decreased in the order waxy>waxy xwx^(a)>wx^(a) x waxy>wx^(a), where waxy is the female in the sample waxyx wx^(a), and wx^(a) is the female in the sample wx^(a) x waxy. In acomparison between wx^(a) and normal starch, the viscosity of cookedstarch increased in the order normal<normal x wx^(a)<wx^(a) xnormal<wx^(a). Thus in these experiments examining the viscosity ofcooked pastes, wx^(a) starch was shown to have a lower viscosity thanwaxy starch and to have a higher viscosity than normal starch. Theelastic properties and gelling abilities of pastes, and the gelproperties of gels produced from this low amylose starch are unknown.Further, the specific mutation resulting in this trait is unknown.

Low amylose barley starches have been shown to contain up toapproximately 5% apparent amylose, though these starches are commonlyreferred to as waxy barley starches (Tester and Morrison, 1992, CerealChemistry 69:654-658). However, this apparent amylose is due to amixture of starch granules in the starch storing organ of the barleyplant. The amylose content of the granules typically ranges from anundetectable level up to approximately 10%, with the granules having thehighest amylose content existing closest to the surface of the seed(Andersson et al, 1999, Journal of Cereal Science 30:165-171). Recentwork with waxy barley starch teaches that starches with amylose contentsup to 6.44% amylose (Li et al, 2001, Food Chemistry 74:407-415) can varyin their viscosity development during cooking under shear (Li et al.,2001, Food chemistry 74:407-415). Of these barley starches with lessthan 6.44% amylose, the starch with no amylose developed viscosity mostrapidly, and those with a higher amylose content were delayed in peakviscosity development. Additionally, all of the waxy barley starchesbegan to develop viscosity at a similar point in the cooking process(time and temperature). No further rheological analysis was conducted onthese starches.

The size and morphology of starch granules and starch molecules producedby a plant of a specific species are characteristic of that species(Jane et al, 1994, Starch/Stärke 46:121-129). Since the physicalproperties of a starch are due to the overall physical composition andstructure of starch granules, exact relationships between one physicalattribute of a starch to the precise cooking behavior of the starch aredifficult to predict. These differences in granules are also accompaniedby species-specific qualities of the lipids contained within the starchgranules (Morrison, 1988, Journal of Cereal Science 8:1-15; Tester andMorrison, 1992, Cereal Chemistry 69:654-658), the species-specificstructure of the amylopectin (Jane et al, 1999, Cereal Chemistry76:629-637), and the species-specific size and structure of the amylose(Takeda et al, 1987, Carbohydrate Research 165:139-145; Hizukuri et al,1981, Carbohydrate Research 94:205-213; Takeda et al, 1989, Cerealchemistry 66:22-25; Takeda et al, 1986, Carbohydrate Research148:299-308; Takeda et al, 1984, Carbohydrate Research 132:83-92). It isequally well known that starch physical behavior is dependent on all ofthese properties (Gidley and Bulpin, 1989, Macromolecules 22:341-346;Eliasson and Kim, 1995, Journal of Rheology 39:1519-1534; Lucinec andThompson, 1998, Cereal Chemistry 75:887-896; Klucinec and Thompson,1999, Cereal Chemistry 76:282-291; Jane et al, 1999, Cereal Chemistry76:629-637; Klucinec and Thompson, 2002a, Cereal Chemistry, 79:19-23;Klucinec and Thompson, 2002b, Cereal Chemistry, 79:24-35). This point isillustrated by the desirability of waxy potato starch with an amylosecontent below 1% over waxy maize starch because of the better heatstable viscosity of the waxy potato starch in some high-temperaturebaking applications (EP1102547).

Because of the differences in the physical structure and composition ofstarches from different plant species, it is difficult to predictwhether a specific relationship observed between the structure orcomposition of a starch and its cooking and rheological properties inone plant species will be observed if the structure and composition arereproduced in another plant species. However, across starches isolatedfrom various plant species the effects of the absence of amylose areclear as a result of comparing waxy starches to normal starches: normalstarches have the ability to form elastic gels while waxy starches formstable viscous pastes. These properties of normal and of waxy starcheshave been recognized in the literature. However, the relationshipbetween the presence/absence of lower amounts of amylose on the gellingand rheological qualities of starches is unclear. Further, theinteraction between the number of different starch enzymes involved instarch biosynthesis remains unclear. Further, few examples of starchwith amylose contents between 1.5% and 15% which are not heterogeneousmixtures of waxy starch and amylose-containing starch exist. Evenfurther, the general value and application of these starches in productshas not bee recognized: they have not been characterized for their pasteand gel properties and how gels of these starches develop from pastes.

Introduction of traits into new plant lines may be accomplished bytraditional breeding practices, a process which is initiated by crossinga plant line with the trait with a target plant line without the trait(a converted line). The crossing, however, also produces an entirely newcombination of genes within the chromosomes of the resultant plantcontaining the trait. Thus, the identity and agronomic characteristicsof the original plant lines are significantly altered. Agronomic traitsare often multigenic and in many plant species these traits are furthercomplicated by multiple sets of chromosomes. Reconstruction of theconverted plant line to its original genetic state but containing thenew trait takes time and a number of crosses, and even after a number ofcrosses the genetics of the converted line will contain some residualfrom the original plant line containing the trait. Thus, the new plantis not equivalent to the unconverted parent with the new trait.

Clearly the corn breeding industry is aware of a method of producingmutations in corn and that these mutations have an effect on theprocessing characteristics of the resultant starch. Chemical mutagenssuch as ethyl methane sulfonate (EMS) produce a mutation in the genome.A method of EMS pollen mutation was published by Neuffer as early as1971 (Neuffer, 1971, Maize Genetics Cooperation Newsletter 45:146-149).EMS mutagenesis may also result in more complex lesions in the plantgenome (Okagaki et al, 1991, Genetics 128:425-431). Another method ofproducing mutations is to use transposon tagging to form a mutation in anucleic acid sequence. This method does not form a point mutation. Thismethod was used by Iowa State University to produce a dominant form ofamylose-extender in corn. At ISU, researchers made a surprisingdiscovery that is evidenced in U.S. Pat. No. 5,004,864 that throughtransposon tagging technology a dominant amylose-extender gene could becreated. The Iowa State researchers' dominant gene produces kernelswithin the 70% apparent amylose region as would be expected. The patentindicates that due to the dominant nature of the gene that in fact theaddition of doses of mutant in the kernel does not increase the level ofapparent amylose produced by the plant. Both processes share theadvantage that the original genetics of the parent are largely retainedafter incorporation of the new trait. A plant which is essentiallyidentical to the parent plant is an isogenic line. Isogenic lines arelines with essentially identical genes. Introducing a new trait into aplant using mutagenesis avoids conventional breeding problems since itdoes not produce an entirely new combination of genes within thechromosomes of the new plant. Though the result of mutagenesis is nearlyisogenic to the parent line with the exception of the introduced trait,the introduction of new traits by mutagenesis is not straightforward.Successful introduction of the trait involves screening thousands ofmutagenized seed for each plant line; fortunately, some traits can beidentified by the phenotype of the starch storing organ (e.g. a seed formaize) and screening can be accelerated.

It is known that the activity and act of enzymes can be altered, notsimply eliminated, through mutagenesis. For example, maize starchsynthase (SS)SSIIa (SEQ ID NO:8) and SSIIb-2 (SEQ ID NO:7) have beensite specific mutagenized (Imparl-Radosevich et al, 1999, FEBS Letters457:357-362; Nichols et al, 2000, Biochemistry 39:7820-7825). Mutantswith much reduced activity or lower affinity for ADPG were obtained(Tables 4 through 6). TABLE 4 Kinetics for SSIIb-2^(a) and mutantsGlycogen as primer Amylopectin as primer Enzyme^(b) V_(max) K_(m)V_(max) K_(m) ADPGIc kinetics^(c) SSIIb-2 118.99 ± 5.06  0.13 ± 0.0274.86 ± 5.46  0.16 ± 0.03 D21N  4.87 ± 0.25 1.48 ± 0.03 2.77 ± 0.26 1.58± 0.11 D21E 13.35 ± 1.32 0.12 ± 0.02 9.30 ± 0.79 0.13 ± 0.03 D139E 25.25± 1.88 0.07 ± 0.02 22.27 ± 2.97  0.09 ± 0.03 E391D 17.16 ± 1.89 1.18 ±0.14 15.05 ± 1.32  1.37 ± 0.14 Primer kinetics^(c) SSIIb-2 97.93 ± 2.970.05 ± 0.01 76.06 ± 3.55  0.16 ± 0.04 D21N  4.31 ± 0.31 0.28 ± 0.03 3.51± 0.69 0.51 ± 0.09 D21E 14.01 ± 0.61 0.21 ± 0.03 9.86 ± 0.49 0.23 ± 0.04D139E 30.51 ± 1.51 0.08 ± 0.01 24.85 ± 1.99  0.07 ± 0.02 E391D 15.05 ±1.32 0.63 ± 0.06 7.44 ± 1.03 0.68 ± 0.07^(a)SSIIb-2 is an N-terminally truncated form of mSSIIb produced in E.coli.^(b)Mutant enzyme designations are based upon the change in the aminoacid sequence. The first letter and number corresponds to an amino acidand its location in the sequence of non-mutant enzyme, respectively, andthe final letter refers to the amino acid replacing the non-mutant aminoacid in the sequence of the mutant enzyme.^(c)V_(max) values are expressed as mol Glucose/min/mg. For ADPGIckinetics, K_(m) values are expressed as mM ADPGIc, glycogenconcentration was 20 mg/ml, and amylopectin concentration was 5 mg/ml.For primer kinetics, K_(m) values are expressed as mg/ml primer. ForSSIIb-2, D21E, and D139E, 1 mM ADPGIc was used, and 5 mM ADPGIc was usedfor D21N and E391D.

TABLE 5 Starch synthase activity of SSIIa mutants as measured in crudeE. Coli extract. Specific activity % activity Enzyme^(a) (nmol/min/mg)of control Wild Type 399 100 R210Q 420 105 R211Q 90 22 R211K 164 41R211E 15 4 H213A 41 10 H213K 36 9 H213W 41 10 H213N 92 23 R214Q 97 24R214K 300 75 R214E 22 6 R221Q 237 59 R269Q 375 94 R284Q 276 69 R492Q 33584 R567Q 423 106^(a)Mutant enzyme designations are based upon the change in the aminoacid sequence. The first letter and number corresponds to an amino acidand its location in the sequence of non-mutant enzyme, respectively, andthe final letter refers to the amino acid replacing the non-mutant aminoacid in the sequence of the mutant enzyme.

TABLE 6 Kinetics for Maize SSlla wild type and mutants^(a) K193R, K193Q,K193E and K497Q Amylopectin as primer Glycogen as primer K_(m) for K_(m)for K_(m) for K_(m for) Enzyme V_(max) ^(b) ADPG^(C) Amylopectin^(d)V_(max) ADPG Glycogen Wild type 49.9 ± 4.06 0.11 ± 0.01 0.20 ± 0.03734.47 ± 1.70 0.12 ± 0.01 0.17 ± 0.03 K193R 18.2 ± 0.91 0.15 ± 0.01 0.525± 0.03  42.27 ± 3.01 0.15 ± 0.02 0.68 ± 0.03 K193Q 17.4 ± 1.58 0.13 ±0.01 0.12 ± 0.008  6.20 ± 0.01 0.10 ± 0.01 0.11 ± 0.02 K193E 10.53 ± 0.50.22 ± 0.01 0.72 ± 0.052 25.45 ± 0.08 0.17 ± 0.02 0.52 ± 0.05 K497Q 13.5± 0.21 5.62 ± 0.23 1.27 ± 0.17  23.8 ± 2.5 6.91 ± 0.33 2.83 ± 0.08^(a)Mutant enzyme designations are based upon the change in the aminoacid sequence. The first letter and number corresponds to an amino acidand its location in the sequence of non-mutant enzyme, respectively, andthe final letter refers to the amino acid replacing the non-mutant aminoacid in the sequence of the mutant enzyme.^(b)V_(max) values are in μmol/min/mg protein.^(c)K_(m) for ADP-glucose (ADPG) are expressed in mM ADP-glucose.^(d)K_(m) for primer (amylopectin and glycogen) are expressed as mg/mlprimer.

From such prior mutagenesis work, it might be expected that mutationscould give rise to an alteration in amylose content. However, based onwhat is known in the literature the invention and finding disclosed andreported herein of a waxy starch with altered rheological propertiescould not be expected.

Known cloning techniques may be used to provide the DNA constructs toproduce an enzyme or protein. Potential donor organisms are screened andidentified. Thereafter there can be two approaches: (a) using enzymepurification and antibody/sequence generation or (b) using cDNAs asheterologous probes to identify the genomic DNAs for enzymes inlibraries from the organism concerned. Gene transformation, plantregeneration and testing protocols are known to the art. In instances inwhich the transgene codes for a starch biosynthetic enzyme it isnecessary to make nucleic acid sequence constructs for transformationwhich also contain regulatory sequences that ensure expression duringstarch formation. These regulatory sequences are present in many grainsand in tubers and roots. For example these regulatory sequences arereadily available in the maize endosperm as DNA encoding StarchSynthases (SS or GBSS) or Branching Enzymes (BE) or other maizeendosperm starch synthesis pathway enzymes. These regulatory sequencesfrom the endosperm ensure protein expression at the correctdevelopmental time (e.g., ADPG pyrophosphorylase).

In the area of polysaccharide enzymes there are reports of vectors forengineering modifications in the starch pathway of plants by use of anumber of starch synthesis genes in various plant species. That GBSSenzymes make amylose is well, known. One specific patent example of theuse of a polysaccharide enzyme shows the use of mutants in GBSS enzymesto modify plant starch. Publications are available, such as WO/9211376,JP04104791, EP788735, WO98/27212, WO02/8052, which teach vectorscontaining DNA to control the activity of GBSS biosynthetic enzymeswithin plant cells. Specifically, these publications refer to thechanges in potato starch due to the introduction of these enzymes. Otherstarch synthesis genes and their use have also been reported to changestarch though generally these are not directly focused on changingamylose content.

Once the ligated DNA which encodes the hybrid polypeptide is formed,then cloning vectors or plasmids are prepared which are capable oftransferring the DNA to a host for expressing the hybrid polypeptides.The recombinant nucleic acid sequence of this invention is inserted intoa convenient cloning vector or plasmid. For starch biosynthetic enzymesthe preferred host is often a starch granule-producing host. However,bacterial hosts can also be employed. Especially useful are bacterialhosts that have been transformed to contain some or all of thestarch-synthesizing genes of a plant. The ordinarily skilled person inthe art understands that the plasmid is tailored to the host. Forexample, in a bacterial host transcriptional regulatory promotersinclude lac, TAC, trp and the like. Additionally, DNA coding for atransit peptide most likely would not be used and a secretory leaderthat is upstream from the structural nucleic acid sequence may be usedto get the polypeptide into the medium. Alternatively, the product isretained in the host and the host is lysed and the product isolated andpurified by starch extraction methods or by binding the material to astarch matrix (or a starch-like matrix such as amylose or amylopectin,glycogen or the like) to extract the product.

The preferred host is a plant and thus the preferred plasmid is adaptedto be useful in a plant. The plasmid should contain a promoter,preferably a promoter adapted to target the expression of the protein inthe starch-containing tissue of the plant. The promoter may be specificfor various tissues such as seeds, roots, tubers and the like; or, itcan be a constitutive promoter for nucleic acid sequence expressionthroughout the tissues of the plant. Well known promoters include the 10kD zein (maize) promoter, the CAB promoter, patatin, 35S and 19Scauliflower mosaic virus promoters (very useful in dicots), thepolyubiquitin promoter (useful in monocots) and enhancements andmodifications thereof known to the art.

The cloning vector may contain coding sequences for a transit peptide todirect the plasmid into the correct location. Coding sequences for othertransit peptides can be used. Transit peptides naturally occurring inthe host to be used are preferred. The purpose of the transit peptide isto target the peptide to the correct intracellular area.

The donor nucleic acid sequence(s) are incorporated into the genome ofthe recipient plant by transformation. Any method suitable for thetarget plant may be employed. Numerous transformation procedures areknown from the literature such as agroinfection using Agrobacteriumtumefaciens or its Ti plasmid, electroporation, microinjection of plantcells and protoplasts, microprojectile transformation, pollen tubetransformation, and “whiskers” technology (U.S. Pat. Nos. 5,302,523 and5,464,765) to mention but a few. Reference may be made to the literaturefor full details of the known methods. The transformed cells may then beregenerated into whole transgenic plants in which the new nuclearmaterial is stably incorporated into the genome. Methods of regeneratingplants are known in the art. Both transformed monocot and dicot plantsmay be obtained in this way, although the latter are usually more easyto regenerate. Once the host is transformed and the proteins expressedtherein, the presence of the DNA encoding the payload polypeptide in thehost is confirmable. The presence of expressed proteins may be confirmedby Western Blot or ELISA or as a result of a change in the plant or thecell.

The present invention provides for generating unexpected and valuabletraits in all recipient plants producing or storing starch. Therecipient plant may be: a cereal such as maize (corn), wheat, rice,sorghum or barley; a fruit-producing species such as banana, apple,tomato or pear; root or tuber crops such as cassaya, potato, yam orturnip; an oilseed crop such as rapeseed, sunflower, oil palm, coconut,linseed or groundnut; a meal crop such as soya, bean or pea; or anyother suitable species. Preferably the recipient plant is of the familyGramineae and most preferably of the species Zea mays.

The present invention provides for methods for generating unexpected andbeneficial traits in all mutant recipient plants producing or storingstarch. The mutant or multiple-mutant recipient plant may be: a cerealsuch as maize (corn), wheat, rice, sorghum or barley; a fruit-producingspecies such as banana, apple, tomato or pear; a root crop such ascassaya, potato, yam or turnip; an oilseed crop such as rapeseed,sunflower, oil palm, coconut, linseed or groundnut; a meal crop such assoya, bean or pea; or any other suitable species. Preferably the mutantor multiple-mutant recipient plant is of the family Gramineae and mostpreferably of the species Zea mays.

The present invention provides methods for generating beneficial traitsin all donor plants producing or storing starch. The donor plant may be:a cereal such as maize (corn), wheat, rice, sorghum or barley; afruit-producing species such as banana, apple, tomato or pear; a rootcrop such as cassaya, potato, yam or turnip; an oilseed crop such asrapeseed, sunflower, oil palm, coconut, linseed or groundnut; a mealcrop such as soya, bean or pea; or any other suitable species.Preferably the mutant or multiple-mutant recipient plant is of thefamily Gramineae and most preferably of the species Zea mays.

The present invention provides methods for generating beneficial traitsin all plants by methods of biotechnology and plant transformation.Those with ordinary skill in the art will recognize that there areseveral ways of affecting amylose content in a plant.

A continuing need to develop starches with improved rheologicalproperties exists. In particular, there is an interest in thedevelopment of starches which as pastes have a high viscosity and havesubstantial elastic character. Such properties would be beneficial infood formulations including pies, puddings, soups, yoghurts, sauces, andother foodstuffs as viscosity builders and suspension aids. Further,such starches could be used for coatings and films in foodstuffs such asbatter coatings. Once deposited on a surface, a paste of the elasticstarch will have a better tendency than existing starches to cling andadhere to a surface rather than flow with gravity.

The present invention provides a starch storing organ which producesstarch which has unique cooking, thickening, and/or gelling properties(herein referred to as an elastic waxy or waxy-E or wx-E starch). Thestarch storing organs of the present invention are characterized asproducing a mutant but active granule-bound starch synthase. The starchof the present invention has a low amylose content. The properties ofthe starches have value in a variety of uses and applications. Thestarch granules described herein were isolated from starch storageorgans from plants which contain a mutant but active granule-boundstarch synthase present in the starch storing organ, have an amylosecontent between 1.5 and 15%, and most preferably an amylose contentbetween 1.5 and 10% and even more preferably an amylose content between2% and 8%, and stain blue or purple with iodine stain.

In the present invention mutagenesis was utilized to developstarch-containing plants which produce a mutant but active granule-boundstarch synthase in starch storage organs and which produce starcheswhich have unique cooking, thickening, and gelling properties and whichalso stain blue with iodine stain and have a low amylose content.Examination of the cooking, thickening, and gelling properties of thestarch recovered from mutant plants was utilized to identify starcheswhich had waxy-E character. Genotypes which showed GBSS activity and alow amylose content are also provided herein. These genotypes may beused to screen for new mutant enzymes or for recombinations of starchsynthesizing enzymes using other methods.

The present invention provides a starch which has unique cooking,thickening, and gelling properties and which also stains blue withiodine stain and has a low amylose content. The present inventionfurther provides a method of producing the disclosed starch. The presentinvention provides methods of producing and identifying useful variantsof plants which produce a starch with a unique functionality and havinga low amylose content as a result of variants of an enzyme. The nucleicacid sequences for any such starch synthesizing enzymes may be used inconstructs according to this invention.

The present invention provides for a starch that has unique cooking,thickening, and/or gelling properties.

The present invention provides a method which results in a plant withthe characteristic of containing a mutation that results in a plant withthe characteristic of containing a mutation that results in reduced butdetectable amylose synthesis when compared to the wild type.

The present invention provides a method which results in a plant withthe characteristic of containing a mutation that results in reduced butdetectable GBSS enzyme activity when compared to the wild type.

The present invention provides a starch that as a result of mutagenesisstains blue or bluish-purple with the application of iodine stain andhas a low amylose content.

The present invention provides a starch from a commercially viable plantline.

The present invention provides a method for crossing a plant producing astarch of the present invention with a second plant producing starch ofthe present invention to produce starch storing organs that produce astarch of the present invention. The resultant propagative structuresfrom said crossing may be grown to produce starch storing organs whichcontain starch of the present invention.

Alternatively, the present invention provides for crossing a plantproducing a starch of the present invention with a waxy plant to producestarch storing organs that produce a starch of the present invention.The resultant propagative structures from the crossing may be grown toproduce starch storing organs which contain starch of the presentinvention.

Additionally, the present invention provides for crossing a plant whichhas at least one plant which produces starch of the present invention inits genetic history with a plant producing starch of the presentinvention or any other plant. The resultant propagative structures maybe grown in a subsequent season to produce starch producing organs whichcontain starch of the present invention.

The present invention also provides for transformation of plants intoplants which produce starch of the present invention.

The present invention provides a starch which after gelatinization orpasting has a higher elastic modulus than pastes of waxy starch andlower than normal starch. In the preferred embodiment, the starch isfrom a maize plant. In other embodiments, the plant is a potato, wheat,rice, or barley plant.

In one embodiment, the starches of the present invention will have atstrains below the yield strain an elastic modulus greater than waxystarch, or preferably an elastic modulus at least two times that of waxystarch, or even more preferably an elastic modulus greater than 10 Pa,or even more preferably an elastic modulus greater than 15 Pa, or mostpreferably an elastic modulus greater than 20 Pa, or further an elasticmodulus between 10 and 100 Pa, or even further between 15 and 60 Pa, orfurther between 20 and 50 Pa when starches of the present invention arecooked as a suspension of 5% starch (dry weight %) in pH 6.5 phosphatebuffer using a Rapid Visco Analyzer 4 and using the instrumentconditions specified in the Newport Scientific Standard 1 Version 5(December 1997) heating and stirring program, and when the resultantpaste is stored for 24 hours at 25° C. before measurement.

In another embodiment, the starches of the present invention will haveat strains below the yield strain an elastic modulus greater than waxystarch, or preferably have an elastic modulus at least two times that ofwaxy starch, or even more preferably an elastic modulus greater than 10Pa, or even more preferably an elastic modulus greater than 15 Pa, ormost preferably an elastic modulus greater than 20 Pa, or further anelastic modulus between 10 and 100 Pa, or even further between 15 and 60Pa, or further between 20 and 50 Pa when starches of the presentinvention are cooked as a suspension in pH 6.5 phosphate buffer using aRapid Visco Analyzer 4 and using the Newport Scientific Standard 1Version 5 (December 1997) heating and stirring program, when theconcentration of the starch is such that a paste of waxy starch at thesame concentration has a final viscosity of between 600 and 850centipoise, and when the resultant paste is stored for 24 hours at 25°C. before measurement.

In a further embodiment, the starches of the present invention whenincorporated into a food product will have at strains below the yieldstrain an elastic modulus greater than the elastic modulus of a productmade with an identical amount of a waxy starch, or preferably an elasticmodulus at least two times that of an identically made and formulatedproduct made with an identical amount of waxy starch, or more preferablyan elastic modulus at least three times that of a product identicallymade and formulated with an identical amount of waxy starch.

In an additional embodiment, the starches of the present invention whenincorporated into a food product will have at strains below the yieldstrain a phase angle less than the phase angle of a product identicallymade and formulated with an identical amount of waxy starch, orpreferably have a phase angle at most 75% that of waxy starch, or evenmore preferably a phase angle less than 15 degrees, or most preferably aphase angle less than 7 degrees.

The present invention provides a starch which after gelatinization orpasting has a higher gel-like character than pastes of waxy starch. Inthe preferred embodiment, the starch is from a maize plant. In otherembodiments, the plant is a potato, wheat, rice, or barley plant.

In another embodiment, starches of the present invention will have atstrains below the yield strain a lower phase angle than waxy starch, orpreferably a phase angle less than 12 degrees, or even more preferably aphase angle less than 10 degrees, or most preferably a phase angle lessthan 6 degrees when starches of the present invention are cooked as asuspension of 5% starch (dry weight %) in pH 6.5 phosphate buffer usinga Rapid Visco Analyzer 4 and using the instrument conditions specifiedin the Newport Scientific Standard 1 Version 5 (December 1997) heatingand stirring program, and when the resultant paste is stored for 24hours at 25° C. before measurement.

In another embodiment, starches of the present invention will have atstrains below the yield strain a lower phase angle than waxy starch, orpreferably have a phase angle less than 12 degrees, or even morepreferably a phase angle less than 10 degrees, or most preferably aphase angle less than 6 degrees when starches of the present inventionare cooked as a suspension in pH 6.5 phosphate buffer using a RapidVisco Analyzer 4 and using the Newport Scientific Standard 1 Version 5(December 1997) heating and stirring program, when the concentration ofthe starch is such that a paste of waxy starch at the same concentrationhas a final viscosity of between 600 and 850 centipoise, and when theresultant paste is stored for 24 hours at 25° C. before measurement.

In one embodiment, starches of the present invention will have atstrains below the yield strain an increase in G′ proportionally lessthan waxy starch with an increase in oscillatory frequency, orpreferably increase less than three fold as the frequency is increasedfrom 0.1 to 100 rad/s oscillatory frequency at a testing strain belowthe yield strain, or even more preferably increase less than 40 Pa asthe frequency is increased from 0.1 to 100 rad/s oscillatory frequencyat a testing strain below the yield strain, or most preferably increaseless than 30 Pa as the frequency is increased from 0.1 to 100 rad/soscillatory frequency at a testing strain below the yield strain, whenstarches of the present invention are cooked as a suspension of 5%starch (dry weight %) in pH 6.5 phosphate buffer using a Rapid ViscoAnalyzer 4 and using the instrument conditions specified in the NewportScientific Standard 1 Version 5 (December 1997) heating and stirringprogram, and when the resultant paste is stored for 24 hours at 25° C.before measurement.

In another embodiment, starches of the present invention will have atstrains below the yield strain an increase in G′ proportionally lessthan waxy starch with an increase in oscillatory frequency, orpreferably increase less than three fold as the frequency is increasedfrom 0.1 to 100 rad/s oscillatory frequency at a testing strain belowthe yield strain, or even more preferably increase less than 40 Pa asthe frequency is increased from 0.1 to 100 rad/s oscillatory frequencyat a testing strain below the yield strain, or most preferably increaseless than 30 Pa as the frequency is increased from 0.1 to 100 rad/soscillatory frequency at a testing strain below the yield strain whenstarches of the present invention are cooked as a suspension in pH 6.5phosphate buffer using a Rapid Visco Analyzer 4 and using the NewportScientific Standard 1 Version 5 (December 1997) heating and stirringprogram, when the concentration of the starch is such that a paste ofwaxy starch at the same concentration has a final viscosity of between600 and 850 centipoise, and when the resultant paste is stored for 24hours at 25° C. before measurement.

In a further embodiment, the starches of the present invention whenincorporated into a food product will at strains below the yield strainhave an oscillatory frequency dependence less than food productsformulated and made identically with waxy starch and have an oscillatoryfrequency dependence greater than or equivalent to normal starch.

The present invention provides a starch which after gelatinization orpasting has a low-temperature stability greater than or equivalent tonormal starch. In the preferred embodiment, the starch is from a maizeplant. In other embodiments, the plant is a potato, wheat, rice, orbarley plant.

In one embodiment, the starches of the present invention have anequivalent or lower differential scanning calorimetry retrogradationenthalpy than normal starch; or preferably a lower differential scanningcalorimetry retrogradation enthalpy than normal starch aftergelatinizing the starch by heating it to 140° C. at 10° C. per min,cooling the starch to 4° C., holding the starch for seven days at 4° C.,and then analyzing the retrogradation enthalpy observed after reheatingthe starch from 5° C. to 140° C. at 10° C. per min; or most preferably adifferential scanning calorimetry retrogradation enthalpy between 3.5J/g and 10 J/g after the starch as a 25% w/w suspension in water hasbeen heated to 140° C. at 10° C. per min, cooled to 4° C., held forseven days at 4° C., and then analyzed by reheating the starch from 5°C. to 140° C. at 10° C. per min.

In an additional embodiment, the starches of the present invention havea lower differential scanning calorimetry amylose-lipid complex enthalpythan normal starch; or preferably have an average amylose-lipid complexenthalpy less than 1.2 J/g and most preferably less than 1.1 J/g.

In a further embodiment, the starches of the present invention havegreater paste stability than normal starch as detected by changes in therheological properties of pastes prepared with the starches of thepresent invention between two storage time points.

The present invention provides a starch which has the ability to formgel structures unlike those of waxy or normal starch. In the preferredembodiment, the starch is from a maize plant. In other embodiments, theplant is a potato, wheat, rice, or barley plant.

In one embodiment, the gelatinized starches of the present inventionhave greater ability to form gels in a range of useful starch contentsthan do waxy starches, or between a range of starch contents between 2and 80%, and preferably in a range of starch contents between 2% and40%, and more preferably in a range of starch contents between 2% and20%, and most preferably in a range of starch contents between 5 and15%.

In another embodiment, the gelatinized starches of the present inventionform easily deformable, highly resilient gel structures rather thanfirm, brittle gel structures formed by normal starch at the sameconcentration, or preferably form gels which do fracture as do normalstarches when they contain 10% gelatinized starch solids, or morepreferably have a resiliency greater than 50% when they contain 10%gelatinized starch solids, or most preferably form gels without adefined fracture point and a firmness below 30 g-s and a resilience ofat least 50% when the starches are cooked as a suspension of 10% starch(dry weight %) using a Rapid Visco Analyzer and the instrumentconditions specified in the Newport Scientific Method 1 (STD1) Version 5method for the instrument and the resultant pastes are stored for sevendays at 4° C. with negligible loss in water.

The present invention provides a starch which has cooking viscositystability higher than waxy starches. In the preferred embodiment, thestarch is from a maize plant. In other embodiments, the plant is apotato, wheat, rice, or barley plant.

In one embodiment, the starch of the present invention developsviscosity at a slower rate than does waxy starch which is cooked underthe same heating and shear conditions, and preferably the time betweenthe pasting time and the peak time is greater than that duration forwaxy starch, and more preferably the time between the pasting time andthe peak time is greater than 90 seconds, and most preferably the timebetween the pasting time and the peak time is greater than 75 secondswhen starches of the present invention are cooked as a suspension of 5%starch (dry weight %) in pH 6.5 phosphate buffer using a Rapid ViscoAnalyzer and the instrument conditions specified in the NewportScientific Standard 1 Version 5 (December 1997) heating and stirringprogram.

In a further embodiment, the starch of the present invention developsviscosity at a slower rate than does waxy starch which is cooked underthe same heating and shear conditions, and preferably the time betweenthe pasting time and the peak time is greater than that duration forwaxy starch, and more preferably the time between the pasting time andthe peak time is greater than 90 seconds, and most preferably the timebetween the pasting time and the peak time is greater than 75 secondswhen starches of the present invention are cooked as a suspension in pH6.5 phosphate buffer using a Rapid Visco Analyzer 4 and using theNewport Scientific Standard 1 Version 5 (December 1997) heating andstirring program, when the concentration of the starch is such that apaste of waxy starch at the same concentration has a final viscosity ofbetween 600 and 850 centipoise.

In another embodiment, the starch of the present invention reaches apeak viscosity at a time later than does waxy starch, preferably thepeak time is greater than four minutes, most preferably the peak time isgreater than five minutes when starches of the present invention arecooked as a suspension of 5% starch (dry weight %) in pH 6.5 phosphatebuffer using a Rapid Visco Analyzer 4 and the instrument conditionsspecified in the Newport Scientific Standard 1 Version 5 (December 1997)heating and stirring program.

In an additional embodiment, the starch of the present invention reachesa peak viscosity at a time later than does waxy starch, preferably thepeak time is greater than four minutes, and most preferably the peaktime is greater than five minutes when starches of the present inventionare cooked at a suspension of in pH 6.5 phosphate buffer using a RapidVisco Analyzer 4 and the instrument conditions specified in the NewportScientific Standard 1 Version 5 (December 1997) heating and stirringprogram when the concentration of the starch is such that a paste ofwaxy starch at the same concentration has a final viscosity of between600 and 850 centipoise.

In another embodiment, the starch of the present invention reaches apeak viscosity at a time later than does waxy starch, most preferablythe peak time is greater than four minutes when starches of the presentinvention are cooked as a suspension of 5% starch (dry weight %) in pH6.5 phosphate buffer using a Rapid Visco Analyzer and the instrumentconditions specified in the Newport Scientific ST-01 Revision 3 heatingand stirring program for the instrument.

In yet another embodiment, the starch of the present invention losesviscosity at a slower rate than does waxy starch after reaching a peakviscosity, preferably the breakdown viscosity to peak viscosity (B/P)ratio is less than 35% and most preferably less than 30% when starchesof the present invention are cooked as a suspension of 5% starch (dryweight %) in pH 6.5 phosphate buffer using a rapid Visco Analyzer andthe instrument conditions specified in the Newport Scientific Standard 1Version 5 (December 1997) heating and stirring program.

In yet another embodiment, the starch of the present invention losesviscosity at a slower rate than does waxy starch after reaching a peakviscosity, preferably the breakdown viscosity to peak viscosity (B/P)ratio is less than 35% and most preferably less than 30% when starchesof the present invention are cooked as a suspension of in pH 6.5phosphate buffer using a Rapid Visco Analyzer 4 and the instrumentconditions specified in the Newport Scientific Standard 1 Version 5(December 1997) heating and stirring program when the concentration ofthe starch is such that a paste of waxy starch at the same concentrationhas a final viscosity of between 600 and 850 centipoise.

In an additional embodiment, the starch of the present inventiondevelops a paste with a higher final viscosity than a paste of waxystarch, preferably the final viscosity is greater than 850 cp and morepreferably is greater than 900 cp when starch of the present inventionis cooked as a suspension of 5% starch (dry weight %) in pH 6.5phosphate buffer using a Rapid Visco Analyzer 4 and the instrumentconditions specified in the Newport Scientific Standard 1 Version 5(December 1997) heating and stirring program, and preferably the finalviscosity is greater than 650 cp and more preferably is greater than 700cp when starches of the present invention are cooked at a suspension of5% starch (dry weight %) in pH 6.5 phosphate buffer using a Rapid ViscoAnalyzer 4 and the instrument conditions specified in the NewportScientific ST-01 Revision 3 heating and stirring program for theinstrument.

The present invention includes the making of sols and pastes of thestarch of the present invention in the presence of dissolved solutes.The present invention includes the making of gels of the starch of thepresent invention. The present invention includes making sols or gels ofthe starch of the present invention for use in foodstuffs such as piefillings, puddings, soups, sauces, gravies, coatings, candies and/orconfectionary products, and/or yoghurts and other dairy products. Thepresent invention includes adding the starch of the present invention asan ingredient to foodstuffs such as pie fillings, puddings, soups,sauces, gravies, coatings, candies and/or confectionary products, and/oryoghurts and other dairy products.

The present invention provides a starch which has an amylose contentbetween waxy starch and normal starch. In the preferred embodiment, thestarch is from a maize plant. In the preferred embodiment, the starchesof the present invention have amylose contents between 1.5% an 15% on aweight basis. In another embodiment, the starches of the presentinvention have amylose contents between 2% and 15% on a weight basis. Inan additional embodiment, the starches of the present invention haveamylose contents between 2.5% and 15% on a weight basis. In anadditional embodiment, the starches of the present invention haveamylose contents between 3.5% and 15% on a weight basis. In a furtherembodiment, the starches of the present invention have amylose contentsbetween 1.5% and 10% on a weight basis. In an additional embodiment, thestarches of the present invention have amylose contents between 2% and10% on a weight basis. In another embodiment, the starches of thepresent invention have amylose contents between 2.5% and 10% on a weightbasis. In a further embodiment, the starches of the present inventionhave amylose contents between 3.5% and 10% on a weight basis. In anotherembodiment, the starches of the present invention have amylose contentsbetween 1.5% and 8% on a weight basis. In a further embodiment, thestarches of the present invention have amylose contents between 2% and8% on a weight basis. In an additional embodiment, the starches of thepresent invention have amylose contents between 2.5% and 8% on a weightbasis. In an additional embodiment, the starches of the presentinvention have amylose contents between 3.5% and 8% on a weight basis.In a further embodiment, the starch is a potato starch with an amylosecontent between 3.5 and 12.5 and more preferably between 4% and 12.5%.In another embodiment, the starch is a wheat starch with an amylosecontent between 1.5% and 15% and more preferably between 2.5% and 15%and most preferably between 3% and 10%. In an additional embodiment, thestarch is a rice starch with an amylose content between 1.5% and 15% andmore preferably between 3% and 15% and most preferably between 3% and6%.

The present invention provides a method of forming a starch of thepresent invention in the starch storing organs of wheat, barley, rice,sorghum, oats, rye or maize. In the preferred embodiment, the starchbearing plant is a maize plant. In other embodiments, the plant is apotato, wheat, rice, or barley plant.

The present invention further provides a starch of the present inventionextracted from the starch producing organs of a plant. In the preferredembodiment, the starch bearing plant is a maize plant. In otherembodiments, the plant is a potato, wheat, rice, or barley plant. In oneembodiment, the starch storing organs are formed on a plant grown from apropagative structure after selection following mutagenesis.

The present invention provides a method of producing a starch of thepresent invention in the starch storage organs of a plant comprising thesteps of: applying EMS to pollen of plants, forming treated pollen;self-pollinating plants with the treated pollen; selecting plantpropagative structures with at least one mutation which appear toproduce starch storage organs containing starch of the presentinvention; planting said plant propagative structures to produceadditional plant propagative structures; selecting propagativestructures from plants which appear to produce starch storage organscontaining starch of the present invention; repeating this cycle ofplanting and selection to increase propagative structure quantities;optionally, said plants may be backcrossed to ensure purity; extractingstarch wherein said starch is a starch of the present invention. Thismethod can include the step of increasing said plant propagativestructures.

The present invention provides a method of producing a starch of thepresent invention in the starch storage organs of a plant comprising thesteps of: mutagenizing propagative structures of plants; growing plantsfrom said propagative structures; selecting plant propagative structureswith at least one mutation which appear to produce starch storage organscontaining starch of the present invention; planting said plantpropagative structures to produce additional plant propagativestructures; selecting propagative structures which appear to producestarch storage organs containing starch of the present invention;repeating this cycle of planting and selection to increase propagativestructure quantities; optionally, said plants may be backcrossed toensure purity; extracting starch wherein said starch is a starch of thepresent invention. This method can include the step of increasing saidplant propagative structures.

The present invention provides a method of producing a starch of thepresent invention in the starch storage organs of a plant comprising thesteps of: mutagenizing cells of plants; regenerating plants from saidcells; selecting plant propagative structures with at least one mutationwhich appear to produce starch storage organs containing starch of thepresent invention; planting said plant propagative structures to produceadditional plant propagative structures; selecting propagativestructures which appear to produce starch storage organs containingstarch of the present invention; repeating this cycle of planting andselection to increase propagative structure quantities; optionally, saidplants may be backcrossed to ensure purity; extracting starch whereinsaid starch is a starch of the present invention. This method caninclude the step of increasing said plant propagative structures.

Additional method steps can be steps of planting said propagativestructures to produce plants with the intent to form more propagativestructures which will produce starch storing organs containing starch ofthe present invention on starch bearing plants, or the step ofharvesting the propagative structures, or the step of crossing thestarch bearing plant with a second plant producing starch of the presentinvention wherein hybrid propagative structures are formed on at leastone of the plants and then the additional step of harvesting thepropagative structures. The present invention's scope also encompassesthe step of harvesting the starch storing organs for the extraction ofstarch.

The present invention can also be described as a method of producingplants which produce starch storing organs which contain starch of thepresent invention including the steps of inducing at least one mutationin the waxy locus of plants; selecting propagative structures from theplant having at least one mutation; growing plants from the propagativestructures; forming propagative structures on the plants; and extractingthe starch of the present invention from the starch storing organs. Moreparticularly the mutation is located in the starch-affecting locus thewaxy locus in the plant's genome, and even more particularly, themutation is a point mutation.

The present invention also includes a product which is a starch of thepresent invention extracted from the starch producing organs of a plantcomprising starch produced by the plant having at least one mutationoriginally induced into the genetic ancestry of the plant by EMS and atleast one of the mutations wherein the starch storing organs of the planproduce a starch of the present invention.

In another embodiment, the starch storing organs are formed by selectionfollowing plant transformation designed to reduce the activity of theGBSS enzyme in a normal plant.

The present invention can also be described as a method of producingplants which produce a starch of the present invention including thesteps of: inducing an antisense construct for a starch affecting locusof the seed bearing plants; selecting propagative structures from theplant having the antisense construct; growing plants from thepropagative structures; forming starch storing organs on the plants; andextracting the starch of the present invention from the starch storingorgans.

The present invention also provides the cDNA encoding a granule boundstarch synthase which has the sequence SEQ ID NO:2.

In yet another embodiment, the starch storing organs are formed byselection following plant transformation designed to increase theactivity of a GBSS-like enzyme in a waxy plant.

The present invention can also be described as a method of producingplants which produce a starch of the present invention including thesteps of: inducing a sense construct for a starch affecting locus of theseed bearing plants; selecting propagative structures from the planthaving the sense construct; growing plants from the propagativestructures; forming starch storing organs on the plants; and extractingthe starch of the present invention from the starch storing organs.

The present invention can also be described as a method of producingplants which produce a starch of the present invention including thesteps of: inducing an expression construct for a starch affecting locusof starch storing plants having a mutation in amylose formation;selecting propagative structures from the plant having the expressionconstruct; growing plants from the propagative structures; formingstarch storing organs on the plants; and extracting starch of thepresent invention from the starch storing organs. The inventionadditionally provides transformed plants containing one or more copiesof the said cDNA in the sense orientation.

The present invention also provides the granule bound starch synthasewhich has the amino acid sequence SEQ ID NO:4.

In further embodiments of this invention, the starch bearing plant maybe any cereal plant (such as wheat, barley, sorghum, rice, oats, rye,etc.) or any starch forming plant. In the preferred embodiment, theplant is a maize plant. In other embodiments, the plant is a potato,wheat, rice, or barley plant.

Broadly the present invention provides for a the starch storing organsof a plant which contain starch which has unique cooking and functionalproperties and starch which has an amylose content below 15% (suchstarches are herein referred to as a waxy-E starch). The presentinvention provides for the use of a potato starch with an amylosecontent between 3.5% and 12.5% and more preferably between 4% and 12.5%to increase the elasticity of a product. The present invention providesfor the use of a wheat starch with an amylose content between 1.5% and15% and more preferably between 2.5% and 15% to increase the elasticityof a product. The present invention provides for the use of a ricestarch with an amylose content between 1.5% and 15% and more preferablybetween 3% and 15%, and most preferably between 3% and 6% to increasethe elasticity of a product. The present invention provides for the useof a barley starch with an amylose content between 1.5% and 15% and morepreferably between 7% and 15% to increase the elasticity of a product.The present invention provides for the use of a corn starch with anamylose content between 1.5% and 15% and more preferably between 2.5%and 15% to increase the elasticity of a product.

Three seed sets EX385wx-E1, EX56wx-E1, and EX12wx-E2 have been depositedas EX385wxa, EX56wxa and EX12wxa, respectively, on Sep. 27, 2001, withthe American Type Culture Collection, 10801 University Blvd., Manassas,Va. 20110-2209, under conditions of the Budapest Treaty, and assignedAccession Nos. PTA-3730, PTA-3731 and PTA-3732, respectively.

The present invention provides, therefore, a plant starch containing areduced amylose content and having an EM greater than, alternatively atleast twice that of, the EM of a waxy starch of the same plant speciesand an EM less than the EM of a starch of a wild type plant of the samespecies, wherein the AP ratio of the plant starch of the invention iswithin 0.5 of the starch of the wild type plant of the same species. Inone embodiment, the plant starch of the present invention has an EM ofat least 10 Pascals and the AP ratio of the plant starch of the presentinvention is within 0.5 of a starch of a wild type plant of the samespecies.

In one embodiment, the starch of the present invention is as describedabove and the EM is measured after the starch has been cooked as asuspension of starch using a Rapid Visco Analyzer 4 instrument, andinstrument conditions specified in the Newport Scientific Method 1(STD1) Version 5 heating and stirring profile, and stored for 24 hoursat 25° C. In a further embodiment, the starch of the present inventionhas a phase angle below the yield strain of less than that of a waxyplant starch of the same plant species.

The starch of the present invention may be further characterized ashaving more of a gel character below the yield strain than a waxy plantstarch of the same plant species and less of a gel character than aplant starch of a wild type plant of the same species.

The starch of the present invention may be also characterized as havingan increase in G′ less than two fold when subjected to a strain of belowthe yield strain, as the oscillatory testing frequency is increased from0.1 to 100 radians per second. Moreover, the plant starch of the presentinvention may have a firmness below 30 g-s and above 1 g-s after beingcooked as a suspension of 10% starch (dry weight %) according to the RVAStandard Method and then stored for seven days at 4° C., and the APratio of the plant starch of the invention is within 0.5 of the AP ratioof starch of a wild type plant of the same species. The plant starch ofthe present invention has a resilience of at least 50% after having beencooked as a suspension of 10% starch (dry weight %) according to the RVAStandard Method and then stored for seven days at 4° C. after cooking,and the AP ratio of the plant starch is within 0.5 of the AP ratio ofstarch of a wild type plant of the same species. The plant starch of thepresent invention demonstrates, according to the RVA Standard Method, atime of greater than 75 seconds between pasting time and peak time afterthe starch has been cooked at a concentration such that the finalviscosity of a waxy starch of the same species cooked at saidconcentration is between 600 and 850 centipoise, the AP ratio of thestarch being within 0.5 of the AP ratio of starch of a wild type plantof the same species.

The plant starch of the present invention contains a reduced amylosecontent and demonstrates a ratio of breakdown viscosity to peakviscosity of less than 35%, as measured by the RVA Standard Method,after the starch has been cooked at a concentration whereby the finalviscosity of a waxy starch of the same species cooked at saidconcentration is between 600 and 850 centipoise, the plant starch havingan AP ratio of within 0.5 of the AP ratio of starch of a wild type plantof the same species.

The starch described herein may be obtained from a plant containing atleast one mutation in the waxy locus of said plant. The plant starch ofthe present invention may be obtained from a plant selected from a cornplant, a potato plant, a wheat plant, a rice plant or a barley plant.

The present invention provides a plant which produces the starch of thepresent invention. The plant of the present invention may have reducedGBSS activity as a result of at least one of a genetic mutation and agenetic transformation.

The present invention provides a method of producing a starch of thepresent invention by a method which includes the steps of applying EMSto pollen of plants, forming treated pollen, pollinating plants with thetreated pollen or propagation structures, harvesting M1 propagativestructures produced from the pollinated plants, planting the M1propagative structures, harvesting M2 propagative structures from theplanted M1 propagative structures, and selecting and/or screening starchfrom the M2 propagative structures. The present invention provides amethod of producing a starch of the present invention which includes thesteps of inducing a mutation in a starch affecting locus of starchstorage organ bearing plants, selecting propagative structures from themutant plants, growing plants from the propagative structures, andselecting and/or screening starch storing organs.

The present invention provides starch selected and/or screened accordingto the methods, such as those described above, disclosed herein.

In one embodiment, the invention provides a method of producing a plantstarch of the invention which includes incorporating a mutation into thegenetic ancestry of said plant, wherein the mutation results in theproduction of the starch. The plants of the present invention may be acorn plant, a potato plant, a wheat plant, a rice plant or a barleyplant. The present invention further provides propagative andnon-propagative parts of the disclosed plants.

The present invention provides an isolated nucleic acid moleculeencoding a polypeptide having the starch synthase activity of apolypeptide having the amino acid sequence of SEQ ID NO:4. A nucleicacid sequence encoding the amino acid sequence of or including SEQ IDNO:4 is provided. An isolated nucleic acid molecule having the nucleicacid sequence of SEQ ID NO:2 is further described herein and provided bythe present invention.

The present invention provides a sol or paste containing the starch ofthe present invention, as well as a foodstuff containing the same. Thepresent invention further provides a gel of the starch of the presentinvention, as well as a foodstuff containing the same. A foodstuffcontaining the starch of the present invention is also provided herein.Methods of making the foodstuffs described herein are also describedherein, such as including the steps of admixing a starch, gel, sol,paste and/or sol of the present invention with edible ingredients.Methods of making starch preparations more elastic are also providedherein which include admixing the presently described starch, gel, sol,paste and/or sol of the present invention with edible and/orstarch-containing ingredients and/or components requiring more elasticproperties.

Still further objects and advantages will become apparent from aconsideration of the ensuing description, accompanying drawings, andexamples.

Starch is the granular or powdery complex carbohydrate that is the chiefstorage form of carbohydrate in plants.

Amylose content is the quantity of amylose in a starch on a dry weightbasis determined by comparison to standards.

Normal starch is the starch extracted from the seed of a plant with theexpected genes regulating the starch biosynthetic pathway (wild types)that consistently averages an amylose content of 18% to 28%. Such normalstarch stains homogeneously blue or purple upon iodine staining.

Waxy starch is the starch extracted from the seed of a plant thatconsistently stains homogeneously red, brown or red-brown upon iodinestaining.

Unmodified starch is starch extracted from seed which has not beenfurther processed with chemicals or enzymes or has not been processedthrough a heating, cooling, pressure or any other physical regime withthe intent to alter the chemical, structural or rheological or texturalproperties of the starch from its original state.

Modified starch is any starch which after extraction from seed has beenprocessed with chemicals or enzymes or has been processed through aheating, cooling, pressure or any other physical regime with the intentto alter the structural or rheological or textural properties of thestarch from its original state after extraction.

Mutant is a description of any biochemical entity (e.g. DNA, RNA,protein, enzyme) which has deviated either in structure or in functionor in expression from normal as a result of a change(s) in DNA sequence.

Mutation is an alteration in the DNA which results in a mutantbiochemical entity.

Mutagenized is any plant tissue treated with a mutagen to induce amutation in the plant DNA.

Waxy Mutant is any plant that produces waxy starch. Such starch stainshomogeneously red, or brown or brown-red upon iodine staining.

Propagative structure: for some plants, this may be the fertilizedripened ovule of a flowering plant containing an embryo and capablenormally of germination to produce a new plant. For other plants, thismay be a short, fleshy, usually underground stem bearing minute scaleleaves each of which bears a bud in its axil and is potentially able toproduce a new plant. Additionally, this may be the often undergroundpart of a seed plant body that originates usually from the hypocotyl,functions as an organ of absorption, aeration, and food storage or as ameans of anchorage and support, and differs from a stem especially inlacking nodes, buds, and leaves. Further, this may be any cutting ortissue which may be regenerated into a new plant. A propagativestructure may be a starch storing organ or starch storage organ of aplant.

Starch storing organ or starch storage organ is a plant structure whichstores starch. This may be a propagative structure of a plant.

Sol or Paste is a fluid colloidal system in which the continuous phaseis a liquid and which is utilized primarily for its viscosity or otherTheological attributes.

Pasting is the process or act of producing a paste or sol.

Gel is a semirigid or rigid colloidal system.

Centipoise or cp is a unit of measure of viscosity equivalent to 1×10⁻³pascal seconds (Pa s).

Peak viscosity is the maximum viscosity a starch paste reaches during aprocess.

Hot Paste Viscosity or HP Viscosity is the viscosity of a starch pasteafter 2.5 minutes at 95° C.

Breakdown is the decrease in the viscosity of a starch paste from itspeak viscosity to some minimum viscosity during a process. The minimumviscosity is observed after the peak viscosity in time.

Final Viscosity is the viscosity of a starch paste at the end of aprocess.

Setback is the increase in viscosity of a starch paste from some minimumviscosity attained during a process to the final viscosity. The minimumviscosity is observed after the peak viscosity in time.

Peak time is the time at which the peak viscosity is attained during aprocess.

Pasting temperature is the temperature at which an initial increase inviscosity is detected during a process.

Pasting time is the time at which an initial increase in viscosity isdetected during a process.

GBSS (granule bound starch synthase) Enzyme Activity or GBSS activity isthe activity of 60 kDa starch synthase enzyme visualized on renaturingPAGE gels and distinguished from other starch synthase enzyme activitiesin that it stains as a dense blue or dark band upon KI/I₂ staining dueto the transfer of glucosyl units from ADP-glucose supplied in thereaction mixture to either glycogen or amylopectin embedded I thepolyacrylamide gel matrix via formation of (1-4) linkages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Iodine staining properties of starch granules. Bulk stainingproperties are to the left and the staining characteristics of arepresentative field of starch granules are to the right. Starch namesare indicated at the far left of the drawing. All waxy starches areidentified by either the “wx” designation (lab-isolated) or by the“Waxy” designation (commercially-isolated). All waxy-E starches aredesignated by the “wx-E1” or “wx-E2” designation.

FIG. 2. High performance size exclusion chromatography of debranchednormal starch and debranched waxy starch from the EX68 background anddebranched EX52wxae starch. Differential refractive index response isplotted against elution time.

FIG. 3. High performance size exclusion chromatography of debranchedwaxy starch and debranched waxy-E starches. The inset figure shows thefull response of the detector. In both drawings, the differentialrefractive index response is plotted against elution time.

FIG. 4. RVA viscograms of 5% starch suspensions in a pH 6.5 buffer witha 2.5 minute cooking step at 95° C. Viscosity and temperature areplotted against time.

FIG. 5. RVA viscograms of 5% starch suspensions in a pH 6.5 buffer witha 20 minute cooking step at 95° C. The inset figure shows the first 500seconds of the analysis to better show the delayed development ofviscosity of waxy-E starches relative to waxy starches. In bothdrawings, viscosity and temperature are plotted against time.

FIG. 6. Frequency dependence (rad/s) at 1% strain of 5% starch pastesprepared in a pH 6.5 buffer using the RVA programmed with a 2.5 minutecooking step at 95° C. Elastic modulus and phase angle are plottedagainst frequency.

FIG. 7. Strain dependence at 1 rad/s frequency of 5% starch pastesprepared in a pH 6.5 buffer using the RVA programmed with a 2.5 minutecooking step at 95° C. Elastic modululs and phase angle are plottedagainst strain (%).

FIG. 8. A high performance anion exchange chromatogram ofisoamylase-debranched EX12wx-E2 starch. Detector response innanocoulombs is plotted against time.

FIG. 9. A comparison of the relative chain length distribution of EX68waxy starch, EX12wx-E2 starch and EX52 waxy amylose-extender doublemutant starch. Relative percent area is plotted against the degree ofpolymerization.

FIG. 10. (a) Detection of starch synthase activities associated withimmature starch granules (14 to 23 days) in renaturing gradient gels (7to 20%). An equal amount of starch (based on fresh weight) was loaded ineach lane. The identity of the starch and the maturity of the seed foreach lane are indicated. (b) Detection of starch synthase activitiesassociated with mature starch granules in renaturing gels (7 to 20%). Anequal amount of starch was loaded in each lane. The identity of thestarch for each lane is indicated.

FIG. 11. (a) Detection of the GBSS protein associated with immaturestarch granules (14 to 23 days) using western blotting. The identity ofthe starch and the maturity of the seed for each lane is indicated. (b)Detection of GBSS protein associated with mature starch granules usingwestern blotting. The identity of the starch and the maturity of theseed for each lane is indicated.

FIG. 12. Detection of the quantity of GBSS protein associated withimmature starch granules (14 to 23 days) using coomassie staining. Theidentity of the starch and the maturity of the seed for each lane isindicated.

FIG. 13. (a) is a schematic showing the design and restriction enzymesites of plant transformation vectors used to alter nucleic acidexpression levels in plants. (b) is a schematic showing the design andrestriction enzyme sites of plant transformation used to introduce annucleic acid sequence into plants.

The invention describes the production of, identification of, andexamination of the starch extracted from plants such as maize plantsand/or other plants which produce waxy-E starch. The waxy-E starch hasseveral characteristics which are in combination an improvement overwaxy starches:

1) The waxy-E starch produces a high peak viscosity and retains moreviscosity at high temperatures under shear than does waxy starch.

2) The waxy-E starch has unique paste and gel Theologicalcharacteristics.

3) The waxy-E starch has useful low-temperature paste and gel stability.

These properties are a result of the unique molecular composition of thewaxy-E starch, primarily that the waxy-E starch has an amylose contentbelow 10% distributed throughout the bulk of the starch granules. Theamylose produced is a result of the reduced but detectable activity ofthe GBSS enzyme in the starch storing organ compared to the relativelyhigh GBSS activity of normal starch storing organs and the undetectableGBSS activity from waxy starch storing organs.

Additionally, the invention also encompasses a method of producing awaxy-E starch in plants through mutagenesis or using biotechnology.

Mutant Plant Generation and Screening

Waxy starch may be extracted from a breeding population of corn, wheat,rice, potato, or other starchy crop having a recessive wx gene. Thepopulation contains the wx gene and selections of modified germplasmwhich are homozygous for the wx gene. The present invention allows theproduction of plants capable of producing waxy-E starch in plant inbredsor varieties. The present invention includes the discovery that theseplants can be made by pollen mutagenesis. This process results in thecreation of point mutants within the plant genome. The waxy-E mutantsproduced herein are allelic mutants. The locus is allelic with the waxylocus. The waxy-E locus when mutagenized results in a distinctivephenotype: a starch which has unique cooking and Theological propertiesand a low amylose content and a starch storing organ which contains apartially active GBSS enzyme. The waxy gene of wild types encodesgranule bound starch synthase (GBSS) enzyme, and waxy-E mutants incommon with waxy mutants have a point mutation in the waxy locus.

The improved crops of the present invention having the above describedcharacteristics may be produced by using the following pollenmutagenesis procedure on elite maize inbreds or on any variety of plantspecies. Additionally other technical approaches that can be envisionedwhich lead to the same waxy-E phenotype, including mutagenesis,biotechnology, and breeding.

The method of producing these elite, agronomically sound, high yielding,waxy-E mutants is a known method called mutagenesis. The process isoutlined in the Neuffer paper Maize Genetic Newsletter 45:146. It shouldbe noted that ethylmethane sulfonate (EMS) is a chemical which includesmutations (a mutagen). Like all mutation processes the act of mutationcan adversely effect the agronomic traits especially yields of theplant. However, the starting germplasm is superior to that in which thelow amylose mutant is usually formed. Thus the overall agronomic traitsof the plant of the present invention are more easily preserved andselected for than the industrial approach of recurrent selection orbackcrossing. Mutations were induced in the inbred line by treatingpollen with EMS in paraffin oil according to the procedure described byNeuffer (1974, Maize Genetic Newsletter 45:146). This treatment wasperformed on a number of inbreds from the various plant genotypes ofcereal. This example will focus on the development of maize waxy-Emutants by this process. This mutagenesis process has been used to makea number of cereal mutants, including waxy and amylose-extender. Theprocess does guarantee the generation of and simple identification of awaxy-E plant. Instead, tens of thousands of seeds from hundreds of plantlines required screening to find putative waxy mutants. Within this setof putative waxy mutants, a second intensive screening was required tofind waxy-E mutants. This second screening included increasing theamount of seed, isolating the starch from the seed, and furtherexamining the cooking properties of the resultant starch, examining theamylose content of the resultant starch, and examining the GBSS enzymeactivity of seed. Only after this second intensive screening could a fewmutants of the putative waxy mutants be classified as producing waxy-Estarch. The waxy-E starches of the present invention cannot be producedin maize through heterozygous combinations of normal (Wx) and mutant(waxy) genes by cross pollinating normal and waxy plants. Additionally,the properties of the low amylose starch cannot be reproduced by mixingstarch from normal plants and waxy plants, resulting in mixtures with anamylose content less than 15%. Recurrent selection and backcrossing, themost common technique for producing waxy lines from pre-existing waxylines, would not be successful in producing waxy-E starches of thepresent invention from existing waxy lines. Additionally, recurrentselection and backcrossing require a number of generations to developthe desired plants whereas the waxy-E starches of the present inventionare generated within a single generation through EMS mutagenesis.

The general steps of the one process to produce plant lines producingwaxy-E starch of the present invention include treating inbred pollen(in this case maize) with EMS. Pollen from an inbred line is placed inEMS in oil. A paint brush is used to apply the pollen on to the silks ofa receptive corn ear. This forms the Mutant-1(M1) seed. Such seeds areharvested, grown, and self-pollinated to produce the Mutant-2(M2)kernels. The resulting M2 kernels are examined visually for the waxyphenotype. This is classically a full, opaque endosperm compared tonormal endosperm.

The next step is an increase of the seed by self pollination. Increasesof the seed may occur over one or multiple generations to obtainquantities of seed sufficient for analysis, starch isolation, or furtherbreeding.

When sufficient seed is available the next step is to cross the putativemutant with a waxy seed phenotype to a waxy mutant inbred to provide acrude verification that in fact the kernel is either a waxy or an waxy-Emutant. A standard waxy mutant or waxy-E or other low amylose mutantinbred is selected. The mutant plant is grown and crossed to thestandard and the hybrid seed is once again visually examined forphenotype. If the mutant is the same as the standard then the kernels onthe hybrid should be consistent with one another for the phenotype. Thistest is used because the mutant gene is recessive.

A sample from the increased seed source is further screened for waxy-Estarch production. This is done by rehydrating one or more kernels inwater (50° C. for 1 day) and then crushing the seed to release thestarch. A sample of crushed endosperm is added to a microscope slid, adrop of water added to the sample, and then a cover slide is placed overthe wet sample. To one edge of the microscope slide, a drop of dilutedstock iodine solution (2 g/L iodine, 20 g/L potassium iodide diluted 10×with water) is added and drawn into the endosperm sample by capillaryaction. The leading edge of the iodine solution under the cover slide isthen examined under the microscope. The observation of blue staininggranules is a positive indication that the mutagenesis resulted in thecreation of a waxy-E event. Starch from seed containing putative waxy-Estarch along with waxy phenotype seed is isolated on a larger scale foradditional examination and characterization.

Transgenic Plant Generation and Screening

There are reports of vectors for engineering modification in the starchpathway by use of a number of starch synthesis genes in various plants.For example, the U.S. Pat. No. 5,349,123 described a vector containingDNA to form glycogen biosynthetic enzymes within plant cells tointroduce changes in the potato starch. The present invention provides astarch storing organ with reduced GBSS activity and a starch with aunique rheology and a low amylose content in plants made possible byalterations at the waxy-E locus either by shuffling, mutagenesis orbiotechnology and/or breeding methods.

In the present invention, the waxy-E locus will be generated using (a)standard recombinant methods, (b) synthetic techniques, or (c)combination of both. The isolated nucleic acid may also be produced by“Shuffling” or synthetic arrangement of part or parts of one or moreallelic forms of the nucleic acid sequence of interest. The waxy locuswill be modified either through point mutations, antisense technology,and/or gene silencing via knockdowns, site directed mutagenesis, RNA, orany other methods known in the art to generate the starch of the presentinvention. These changes will reduce/silence the level of expression ofthe waxy gene and/or change its functional properties and thereby willeither reduce the corresponding GBSS protein levels, and/or decrease theactivity of GBSS enzyme.

In some embodiments, the desired and modified polynucleotide of Waxylocus of the present invention with multiple functionalities will becloned, amplified or constructed from any starch producing plant. Theisolated nucleic acid compositions of this invention, such as RNA, DNA,and genomic DNA can be obtained from plants or other biological sourcesusing any number of cloning techniques known in the art. Functionalfragments from different species included in the invention can beobtained using primers (12 to 200 bases) that selectively hybridizeunder stringent conditions. Functional fragments can be identified usinga variety of techniques such as restriction analysis, Southern analysis,primer extension analysis and DNA sequence analysis. Variants includedin the invention may contain individual substitutions, deletions oradditions to the nucleic acid or polypeptide sequence. Such changes willalter, add or delete a single amino acid or a small percentage of aminoacids in the encoded sequence.

Preferred nucleic acid molecules of this invention comprise DNA encodingthe waxy-E locus with a modification introduced in the functionality ofthe GBSS enzyme via any or above said methods from any organism andcomprise nucleic acid sequences set forth hereof (SEQ ID NO:2).

A polynucleotide of the present invention can be attached to a vector,adapter, promoter, transit peptide or linker for cloning and or for itsexpression. Preferred plasmids of this invention are adapted for usewith specific hosts. A polynucleotide of the present invention can beexpressed in sense or antisense orientation (see attached examples formaize, FIG. 13). Plasmids comprising a promoter, a plastid-targetingsequence, a nucleic acid sequence encoding the modified Waxy locus witha modified functionality of GBSS enzyme and a terminator sequence areprovided herein. (Such plasmids are suitable for insertion of DNAsequences encoding the eGBSS enzyme with modified functionality toexpress in selected hosts and produce EM (elastic modulus) starch. Theinvention includes plasmids comprising promoters adapted for bothprokaryotic and eukaryotic hosts. The said promoters may also bespecifically adapted for expression in monocots or in dicots.

Additional sequences may be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art.

The DNA Construct for expressing the waxy-E locus within the host,broadly is as follows:

*optional components

As is known in the art, a promoter is a region of DNA controllingtranscription. Different types of promoters will be selected fordifferent hosts. Lac and T7 promoters work well in prokaryotes, the 35SCaMV promoter works well in dicots, and the polyubiquitin promoter workswell in many monocots. Other suitable promoters include maize 10 kDaZein promoter, GBSS promoter, ST1 promoter, TR1 promoter, napin promoteretc. Any number of different promoters are known to the art and can beused within the scope of this invention. It can be constitutive,inducible, tissue specific and may be homologous or heterologous to thesaid plant.

Also, as is known to the art, an intron is a nucleotide sequence in anucleic acid sequence that does not code for the gene product. Onecomponent of an intron that often increases expression in monocots isthe Adhl intron. This component of the construct is optional.

The transit peptide-coding region is a nucleotide sequence that encodesfor the translocation of the protein into organelles such as plastidsand mitochondria. A transit peptide that is recognized and compatiblewith the host in which the transit peptide is employed is preferred. Inthis invention the plastid of choice is the amyloplast. An example isthe ferredoxin transit peptide.

It is preferred that the hybrid polypeptide be located within theamyloplast in cells such as plant cells that synthesize and store starchin amyloplasts. If the host is a bacterial or other cell that does notcontain an amyloplast, there need not be a transit peptide-codingregion.

A terminator is a DNA sequence that terminates the transcription.

The polypeptides generated by the above method may also includepost-translational modifications known to the art such as glycosylation,acylation, and other modifications not interfering with the desiredactivity of the polypeptide.

A variety of methods are known in the art to transform crops or otherhost cells and any method that provides for efficienttransformation/transfection may be employed. A DNA construct of thepresent invention may be introduced directly into the genomic DNA of theplant cell using techniques such as electroporation, particlebombardment, silicon fiber delivery, or microinjection of plant cellprotoplasts or embryogenic callus. Also, the DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector.

The present invention provides methods for increasing or decreasing theconcentration or composition of Waxy locus that encodes for GBSS enzymein a plant or part thereof. The method comprises transformation of a wxplant cell with an expression cassette comprising waxy-E polynucleotideto obtain a transformed plant cell later on grown to a plant underfavorable growth conditions and the plant expresses the modified GBSSprotein for considerable period of time and it results in production ofa starch of the present invention. The plant cell or the plant partcomprising the isolated nucleic acid is selected by means known to theskilled art, and include Southern blot, DNA sequencing, or PCR analysisusing primers specific to the promoter and to the nucleic acid anddetecting amplicons produced there from. Proteins of the presentinvention are derived from native GBSS by addition or substitution ofone or more amino acids at one or more sites either by geneticpolymorphism or synthetic manipulations known in the art. The protein ofthe present invention can be expressed in a recombinant engineered cellsuch as bacteria, yeast, insect, and plant cells. The proteins of theinvention may be purified using the methods known in the art. Detectionof the proteins that are expressed will be achieved by the methods knownin the art and include, for example radioactive assays,radioimmunoassay, different electrophoresis techniques, western blottingtechnique or immunoprecipitation, enzyme-linked immunosorbent assays(ELISAs), immunofluorescent assays, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), and etc.

Starch Isolation

Starch may be isolated on larger or smaller scales as needed; suchprocedures are well-described in the literature (e.g. Singh et al.,1997, Cereal Chemistry 74: 40-48). All waxy-E starches are easilyisolated in quantity using the above method and the yield lossestypically observed with other single mutants (e.g. amylose-extender,sugary-2, dull) and most especially double mutants are not observed.Additionally, by conducting the initial rehydration of the seed at 50°C., the isolation procedure is able to provide evidence that the waxy-Estarch may be isolated from waxy-E seed using existing processingtechnologies, which often involve an initial rehydration of the seedbetween 50° C. and 55° C.

Starch Amylase Content Analysis

The amylose content of the putative waxy-E starch is determined. Thetest is based on the fact that two polysaccharide components existing instarch form helical polyiodide complexes with differentspectrophotometric properties: the linear amylose complexes iodine toform a deep blue complex and the branched, short chain amylopectinweakly complexes iodine and gives a red coloration (Bailey and Whelan,1961, The Journal of Biological Chemistry, 236:969-973; Banks et al,1971, Carbohydrate Research 17:25-33). Thus, waxy starches aredifferentiated from other starches by their inability to form this deepblue complex when examined in the presence of a commonly used solutionof iodine and potassium iodide. The waxy-E starches contain amylose andthus form this blue complex.

More rigorous determination of the amylose content of waxy-E starches,normal starches, and/or waxy starches may be done through calculation bycomparing the spectrophotometric absorbance of an iodine stained sampleto standards of iodine stained amylose and/or amylopectin and/or waxystarch. The amylose content of the starch is determined from an equationderived from the standard curves for amylose and waxy maize starch(absorbance at 635 nm vs. carbohydrate concentration) and from the totalcarbohydrate of each unknown solution, as determined using the method ofDubois et al (1956, Analytical Chemistry 28: 350-356). A similarstandardization was utilized by Knutson and Grove (1994, CerealChemistry 71: 469-471) to correct the amylose content of the starchbased on the total carbohydrate content of the solution measured. Thus,a weight basis amylose content is obtained.

The waxy-E starches of the present invention have an amylose contentranging from 1.5% to between 8% and 12%, depending on whether or not theabsorbance of the sample is corrected for the small absorbance of theamylopectin of the sample.

Starch Physical Property Analysis

The waxy-E starches of the present invention are confirmed by testingthem against waxy and/or normal and/or other starches for their physicalproperties using several instrumental techniques. These instruments andtechniques, in addition to others, can be used to quantitatively assessthe differences one starch has in relation to another. Hence, some valueof a starch relative to others in food or industrial applications may beassessed and determined. These techniques apply to both unmodifiedstarch or modified starch, with the starch modified using practices andtechniques familiar to those proficient in the art (Whistler, R. L. andBeMiller, J. N. 1997. Carbohydrate chemistry for Food Scientists, EaganPress, St. Paul, pp. 137-150; Whistler and Daniel, 1985, Carbohydrates,in Food Chemistry, O. R. Fennema, ed., Marcel Dekker, Inc., New York,pp. 118-121; Zheng, G. H. et al, 1999, Cereal Chemistry 76:182-188;Reddy and Seib, 2000, Journal of Cereal Science 31:25-39) to improve orchange the physical behavior or chemical structure of the starch.

All of these instrumental techniques require knowledge of the dry solidscontent of the starch. The solids content is calculated by determiningthe percentage moisture of the starch and then subtracting this valuefrom 100. The moisture content of the starch is assessed using theone-stage moisture determination method of Standard Method 44-15A of theAmerican Association of Cereal Chemists (2000, Method 44-15A,Moisture-Air Oven Methods, Approved Methods of the American Associationof Cereal Chemists, Tenth Ed., American Association of Cereal Chemists,Inc., St. Paul, Minn.).

A differential scanning calorimeter (DSC) has the ability to measure orcalculate the quantity of energy (as heat) required to dissociate thestructures holding starch granules together. Sometimes, this heat energyis determined mathematically from a temperature difference. This processof dissociation, called gelatinization, is endothermic (i.e. requiringthe input of energy). Normal starches undergo two thermal transitionsduring gelatinization: one transition at a lower temperature isattributed to the disruption of order between starch chains(dissociation of starch crystallites and unwinding of starch doublehelices) and a higher temperature transition attributed to thedissociation of amylose-lipid complexes. The amylose-lipid transition isnot observed for waxy starches as they have no amylose and containlittle lipid. A DSC has the ability to measure or calculate the quantityof energy (as heat) required to re-dissociate a retrograded starch pastewhich has partially reorganized into double-helical and crystallinestructures. This energy (enthalpy) involved is a measure of starchstability and may vary depending on the solids content of the starch,the storage temperature, and the aqueous environment of the starch. Thegelatinization temperature range and enthalpy of the starch component ofwaxy-E starch is indistinguishable from the gelatinization of the starchcomponent of either waxy or normal starch. I some cases, anamylose-lipid dissociation endotherm is observed, however the magnitudeof this endotherm (as the enthalpy) is significantly smaller thanobserved for normal starch. These results provide additional evidencethat the waxy-E starch may be isolated from waxy-E seed using existingprocessing technologies, which often involve heating steps exceeding 50°C. The retrogradation temperature range of waxy-E starch isindistinguishable from the retrogradation temperature range of eitherwaxy or normal starch. However, the retrogradation enthalpy may beobserved to be between waxy and normal starch though this largelydepends on the starch concentration and the temperature to which thestarch was heated during gelatinization: higher heating temperaturesduring gelatinization result in lower starch retrogradation enthalpiesmore than with lower heating temperatures (Liu, Q. and Thompson, D. B.,1998, Carbohydrate Research 314:221-235). Retrogradation enthalpies ofwaxy-E starches will approach those of waxy starches with increasingcooking temperature, decreasing starch content, or in general,increasing destructurization of the starch granules.

Assessment of the rheology of starch during gelatinization and aftergelatinization are commonly conducted using varying types ofinstruments. Gelatinization of starch granules into pastes is commonlymonitored by continuously shearing the sample as the sample is heatedand cooled in a controlled manner. One instrument which does this iscalled a Rapid Visco Analyzer (RVA; Rapid Visco Analyser 4, NewportScientific Pty. Ltd., Warriewood NSW, Australia). Rapid visco analysisof starch suspensions may be performed using a variety of standardheating and cooing protocols provided with the instrument (Anonymous.1998. Ch. 7, General applications, in the Applications Manual for theRapid Visco Analyzer, Newport Scientific Pty. Ltd., Warriewood NSW,Australia, p. 36) or programmed for specific heating, cooling, and shearrate regimes. The waxy-E starches begin to develop viscosity at slightlyhigher temperatures than waxy starch during initial heating. The waxy-Estarches also develop a peak viscosity at later times than waxy starch,and retain more of the developed viscosity than does waxy starch. Allwaxy-E starches develop a higher peak viscosity than normal starchwithin useful concentrations of starch solids. Finally, waxy-E starchesdevelop higher viscosity pastes than waxy starch as the paste is cooled.Thus, more of the viscosity developed by the waxy-E starch is retainedduring cooking and is reestablished after cooking than is observed forwaxy starch. As the retention of viscosity and improvement of starchstability to heat and shear is often a primary reason for starchchemical modification, the present invention may be viewed as a naturalalternative to chemically modified starches or as a feedstock forimproved chemically modified starches. Such starches could also be usedin countries where some chemically modified food starches are prohibitedby law.

In addition to shear viscosity measurements, more complex assessment ofthe rheological quality of a starch paste may be conducted using anoscillatory shear rheometer which is able to probe the elastic andviscous nature of viscous pastes or gels. These descriptors and theirderivation are described in detail by Biliaderis (1992, Characterizationof starch networks by small strain dynamic rheometry, in Developments inCarbohydrate Chemistry, R. J. Alexander and H. F. Zobel, eds. AmericanAssociation of Cereal Chemists, St. Paul). Generally, a force deformingan object can be divided into two parts: one part which is lost to thematerial during deformation, and one part which is retained by thesample and returned when the deforming force is removed. When normalizedfor the area over which the force is applied and normalized for thestrain (the amount the sample is deformed relative to the thickness orheight of a sample), the total force is termed the “complex modulus”(abbreviated G*), the elastic, conserved, force is termed the “storagemodulus” (abbreviated G′), and the lost force is termed the “viscousmodulus” (abbreviated G″). The relationship between G*, G′, and G″ is:G*=√{square root over (G′ ² +G″ ² )}

Above a given strain, the ability of a starch paste to store the forceapplied to it decreases due to an decrease in long-term interactions(longer than the time taken per deformation cycle) between starchmolecules; the strain at which this begins to occur is termed the “yieldstrain” of the material. For strong biopolymer gels, like amylose, theyield strain is often below 1% (Clark, A. H and Ross-Murphy, S. B.,1987, Advances in Polymer Science 83:57-192). Below the yield strain,the G*, G′, and G″ remain relatively constant, a small, incrementalincrease or decrease in the strain applied to the material will have noeffect on these values. Changes in the paste with strain may also beevaluated by observing the phase angle of the material. The phase anglerepresents how close the material is to a perfectly elastic material(with a phase angle of 0 degrees) or to a perfectly viscous liquid (witha phase angle of 90 degrees). As the strain on a sample increases beyondthe yield strain of a material, the phase angle of a material increasesdue to a decrease in the long-term structure of the material, i.e., thelinks between molecules break under high strain. The frequencydependence of the elastic modulus and viscous modulus may also beexamined with a rheometer. The dependence of the elastic and viscousmoduli on measurement frequency is an indication of the nature of thepolymer system: strong frequency dependence indicates that the system ismore related to a disperson of randomly-interacting polysaccharidemolecules with low long-term order and low gel-like character, whileweak frequency dependence indicates that the structure within the systemis relatively fixed which is a characteristic of polymer gels with highgel-like character. Thus, using continuous shear measurements andoscillatory rheological measurements, the quality of starch pastes maybe assessed. Below the yield strain, waxy-E starch pastes have a higherG′ than waxy starch. Additionally, below the yield strain the lowerphase angles of waxy-E starch pastes compared to waxy pastes indicatesthat the waxy-E pastes have a higher degree of elastic character.Further, the waxy-E starch pastes have a lower frequency dependence thando pastes of waxy starch, indicating that the waxy-E pastes have ahigher gel-like character than do waxy starch pastes. A result of thesecharacteristics is an indication of the behavior of the waxy-E starchwhen at rest or under low deformations: waxy-E starches have a lowerlikelihood to flow under their own weight and produce pastes which arenot as noticeably stringy and cohesive as are those of waxy starch.

Assessment of starch gel characteristics may be examined usingpenetrometers and texture analyzers which have the ability to penetrateor withdraw a probe from a solid or semi-solid sample and measure theforce required to move the probe a given distance. The waxy-E starchesform weak gels at relatively low concentrations (10% w/w starch).Further, gels of waxy-E starch are unique: they do not fracture and theyreturn to their original shape to a large extent after the deformation.A measure of this return to the original shape is the resilience (orresiliency) of the gel, is calculated as the ratio of the positive forceduring probe withdrawal to the positive force during probe penetration(the firmness). Normal starch gels do not exhibit these qualities: theyset into firm gels which fracture under low deformations and remaindeformed after the force acting on the gel has been removed.

The physical properties and benefits of starches are also commonlytested against other starches in applied situations, normally as part ofa food or industrial formulation. Often, starches behave differentlywhen in the presence of dissolved solutes and other materials such asproteins and lipids. Tests in formulations help to confirm the benefitsof a particular starch. From examination of the waxy-E starches in alemon pie filling formulation, the viscosity and rheological propertiesof the waxy-E starches are found to be consistent with those propertiesobserved with the isolated starch in pastes. The similarity in theTheological properties of the fillings after they have been stored forone day and one week additionally shows that the waxy-E starches haveuseful low temperature stability.

Starch Chemical Structure Analysis

Starches may also be differentiated and evaluated based on thestructures of the molecules. One way to assess the differences betweenthe physical structures of starch molecules is by examining thedistribution of the lengths of their chains. This distribution iscommonly assessed using high performance gel permeation chromatography(GPC) (Klucinec and Thompson, 1998, Cereal Chemistry 75:887-896) or highperformance anion exchange chromatography (HPAEC) with pulsedamperometric detection (PA) (Jane et al, 1999, Cereal Chemistry76:629-637). Other chromatographic or similarly functioning methods ofdetection are known to those ordinarily familiar with the art. It iswell known that additional mutations in the starch biosynthetic pathwayaffect the structure of the starch molecules, and that the changes inthe structure of the starch molecules due to these additional mutationsaffect the physical properties of the starch (Jane and Chen, 1992,Cereal Chemistry 69:60-65; Jane et al., 1999, Cereal Chemistry,76:629-637; Klucinec and Thompson, 1998, Cereal Chemistry 75:887-896;Klucinec and Thompson, 1999, Cereal Chemistry 76:282-291; Klucinec andThompson, 2001a, Cereal Chemistry, accepted; Klucinec and Thompson,2001b, Cereal chemistry, accepted). Using HPAEC-PAD, no differences areobserved between the debranched waxy-E starches, debranched waxy starch,or debranched normal starch in their chain length distribution up to adegree of polymerization of 50 glucose units. This result indicates thatalteration of the shorter chains of the waxy-E starch are notresponsible for its unique physical behavior, unlike waxyamylose-extender starch which has an altered distribution of shorterchains.

GPC may also be used to assess the amylose content of the starch sinceamylose molecules are typically longer in length than those ofamylopectin, and GPC detection and separation methods are commonlyoptimized to examine such chain lengths. HPAEC-PAD chromatographymethods are typically insensitive to chains longer than 50 to 100glucose units in length due to the limits of the detection method. Usinghigh performance GPC with a chromatography column set chosen for theexamination of longer starch chains, debranched waxy starches areobserved to elute between 43 minutes and 53 minutes, while debranchednormal starch elutes between 30 minutes and 53 minutes. The differencein the initial elution time of debranched waxy starch and debranchednormal starch is due to the amylose of the normal starch, which elutesbetween 30 and 43 minutes. Debranched waxy-E starches have a low butreproducible amount of starch chains which elute before 43 minutes,indicating that they contain long chains not present in the waxystarches. This long chain material is approximately 24%-28% of the totalmass for normal starch and for waxy-E starches ranges from 1.5% to about8% of the total carbohydrate mass. The long chain material is believedto be amylose. The presence and quantity of this long chain material isconsistent with the spectrophotometric amylose content measurementsdescribed earlier by iodine staining.

Plant Hybrid Production

The production of a hybrid plant involves combining the genetics of atleast two inbred plants. The development of a hybrid corn varietyinvolves three steps: (1) the selection of plants from various germplasmpools; (2) the selfing of the selected plants for several generations toproduce a series of inbred lines, which, although different from eachother, breed true and are highly uniform; and (3) crossing the selectedinbred lines with unrelated inbred lines to produce the hybrid progeny(F1). During the inbreeding process in corn, the vigor of the linesdecreases. Vigor is restored when two unrelated inbred lines are crossedto produce the hybrid progeny. An important consequence of thehomozygous and homogeneous nature of the inbred lines is that the hybridbetween any two specific inbreds will always be the same. Once theinbreds that give a superior hybrid have been identified, the hybridseed can be reproduced indefinitely as long as the homogeneity of theinbred parents is maintained. The inbred mutant of the present inventionis recessive. The recessive nature of the gene makes it necessary in theproduction of hybrid crops such as maize and hybrid wheat to produce theevent in two inbreds. Alternatively, the hybrid crop may be made withanother plant such that the mutant of the present invention is dominantor semi-dominant to those traits contained in the other parent plant.These two inbreds should be suitably crossed to get a hybrid that ishigh yielding and has acceptable commercial agronomic characteristics.For example but not as a limitation in maize one inbred could be fromthe stiff stalk family such as B73 and the other could be a Mo17 orother Lancaster type. Likewise breeders with ordinary skill in the artof plant breeding can select the elite lines that should be mutagenizedto make an acceptable hybrid cross. Alternatively the inbreds inexisting commercial inbred waxy lines can be used to form two newhybrids each containing the desired mutant. In this case the waxy-Eproperties of the hybrid will be midway between the amylose contents ofthe two inbreds. The inbreds containing the desired physical andstructural traits of a low amylose starch can be selected and crossedwith another inbred having the same mutations, different mutations, oradditional mutations to make the hybrid. Other alternative breedingmethods can be used with the inbred to form hybrids or breedingpopulations.

This invention is directed primarily at alteration of starch in grain ofplants using genes from donor species of plants. Alternatively theinvention can be used to alter starch of other recipient plants usinggenes from monocotyledonous plants. This can be achieved by mutagenesisor breeding or a variety of known techniques which are known in the artas genetic engineering. Making hybrid proteins with different glucanchain-extending properties is also possible.

EXAMPLES Example 1 Starch Isolation

Starch is isolated from maize seeds based on the following proceduremodified from Eckhoff et al (1996, Cereal Chemistry 73: 54-57) and Singet al (1997, Cereal Chemistry 74:40-48). Corn kernels (100 g) are mixedwith 200 mL of an aqueous steeping solution (0.3% sodium metabisulfiteand 0.5% lactic acid) in a flask. The flask is then stoppered and themixture then held at 50° C. for 48 hours. After 48 hours the corn isrinsed once with water, transferred to the unmodified 64 fluid oz. Jarof a commercial blender (Vita-Mix Commercial Food Preparing Machine,model VM0101, Vita-Mix Corp., Cleveland, Ohio) along with 150 mL ofwater, and then ground at variable speed setting “5” for 4 minutes, witha pause for 10 seconds every minute to improve homogenization. Theground sample is transferred to a #7 mesh sieve snugly fit atop a 4 Lbucket (Encore Plastics Corp., Byesville, Ohio). Additional starch isremoved from the sample atop the mesh by rinsing it with 150 mL ofwater. The entire assembly is then shaken for 5 minutes using a sieveshaker (CSC-Meinzer sieve shaker, Model 184800-000, CSC ScientificCompany, Inc., Fairfax, Va.) on setting “9” and to which 100V isdelivered through the use of a variable transformer (Powerstat variabletransformer, 117C series, Superior Electric, Bristol, Conn.). Duringshaking an additional 200 mL of water is added to rinse the sample. Thematerial passing through the sieve is returned to the blender jar andthen ground at variable speed setting “938 for 2 minutes, with a pausefor 5 seconds every 30 seconds to improve homogenization. The resultantsample is allowed to settle for 10 minutes, after which time 250-300 mLof liquid is decanted. The remainder of the sample is transferred to a200 mesh sieve snugly fit atop a 4L bucket. The entire assembly is thenshaken for 5 minutes using a sieve shaker as before, during which timethe sample is washed with the previously decanted liquid and anadditional 600 mL of water. The material passing through the sieve isallowed to settle for at least 1.5 hours, at which time most of theliquid is first decanted from the sample and a portion of which is thenreturned to the sample to bring the specific gravity to 1.040-1.045. Thestarch-protein slurry is pumped at 55 mL/min along a 2 inch×96 inch(W×L) aluminum channel placed at a 0.0104 ratio of rise to run. Thedecanted liquid is pumped along the table immediately following thestarch-protein slurry, followed by 125 mL of water. Yellow coloredprotein impurities are moved along the table, when necessary, during thetime when the decanted liquid and 125 mL of water are pumped along thetable by using air squeezed from an empty wash bottle. The starch, asthe white residue remaining on first 90 inches of the table, is allowedto air dry on the table for at least 18 hours and is then scraped fromthe table into a plastic weighing dish. The starch in the dish is driedfor an additional 18 hours at 30° C. in a forced air convection oven,after which it is ground into a fine powder using a retail coffeegrinder and then transferred into a storage bottle. Starch was isolatedon larger scales according to Singh et al (1997, Cereal Chemistry 74:40-48) as needed.

Example 2 Starch Morphology and Color

This experiment was conducted to illustrate the effect of the lowamylose content of waxy-E starch on the iodine staining properties ofthe waxy-E starch.

The waxy-E starch granules extracted as described in Experiment 1 werestudied microscopically and compared to known waxy and normal starches.Additionally, commercial normal maize (Cerestar-USA, C*Gel 03420) andwaxy maize starch (Cerestar-USA, C*Gel 04230) samples were alsoexamined. Light microscope studies showed all of the starches wereshaped similarly and all were highly birefringent under cross-polarizedlight. The iodine staining of the starches is tested by suspending 15 mgof starch in 2.85 mL of water. A stock iodine-iodide solution (2 g/Liodine, 20 g/L potassium iodide) is diluted 100-fold in water. Analiquot (0.15 mL) of the diluted iodine-iodide stock solution is addedto the starch suspension. The starch color was visually examined (FIG.1). All waxy-E starches stained a dark bluish-purple with the additionof iodine stain and could not be distinguished from normal starches.Only the waxy starches stained a light reddish-brown color. Theuniformity of the coloration across starch granules was examined using alight microscope after the addition of three additional 0.15 mL aliquotsof diluted iodine-iodide stock. When the suspensions in FIG. 1 wereexamined under the microscope, the waxy-E starches stained predominantlybluish-purple and could not be distinguished from normal starch by theiriodine staining character (FIG. 1). The commercial waxy starch could beclearly differentiated from the lab-isolated waxy starches by itscontamination with normal starch. For the commercial waxy starch sampleevery microscope field contained one or two bluish-purple stainingstarch granules.

Example 3 Amylose Content & Amylopectin Chain Ratio

These tests were conducted to demonstrate that waxy-E starches may bedifferentiated from waxy and normal starches using two amylosequantitation techniques: iodine binding and gel permeationchromatography.

The amylose content of the starches is tested using an adaptation of themethod of Morrison and Laingnelet (1983, Journal of Cereal Science1:9-20). Starch granules (8 mg) in a microcentrifuge tube are dispersedin 0.4 mL of 90% dimethyl sulfoxide by heating in a boiling water bathfor 1 hour. Samples are agitated every 10 minutes during heating. Thedispersed starch is precipitated by adding 1.6 mL of ethanol andcentrifuged at 3000×g for 5 minutes in a microcentrifuge at roomtemperature. The supernatant material is discarded. The starch pellet iswashed twice with 1.0 mL of ethanol and once with 1.0 mL of acetone,centrifuging the sample as described above each time. The non-granularstarch, free of native lipids which interfere with amylosedetermination, is allowed to dry in the uncapped microcentrifuge tubefor at least two hours. After drying, the non-granular starch isdispersed in 1.0 mL of a solution of 10% 6M urea and 90% dimethylsulfoxide by heating in a boiling water bath for 1 hour. Samples areagitated every 10 minutes during heating. The dispersed sample (0.05 mL)is mixed with 10 mL of water and 0.2 mL of an iodine-iodide solution (2g/L iodine, 20 g/L potassium iodide). Blank solutions withoutcarbohydrate were prepared in the same manner. Normal corn amylosestandards (0.05 mL of 1, 2, 4, 6, and 8 mg/mL stock solutions) were madefrom lab-isolated amylose of at least 95% purity using the method ofKlucinec and Thompson (1998, Cereal Chemistry 75: 887-896). The purityof the amylose was confirmed using the gel permeation chromatographymethod of Klucinec and Thompson (1998, Cereal Chemistry 75: 887-896).Waxy maize, isolated from a known GBSS-absent waxy (null) mutant, wasused for an amylopectin standard [0.05 mL of 2, 4, 6, and 8 mg/mL stocksolutions in addition to 0.1 mL of the 6 mg/mL stock solution (12mg/mL), and 0.1, 0.15, and 0.2 mL of the 8 mg/mL stock solution (16, 24,and 32 mg/mL, respectively)]. The additional DMSO in the 16, 24, and 32mg/mL amylopectin standards has no effect on the linearity of thesubsequently constructed standard curve. The spectrophotometer is zeroedat 635 nm with the blank solution, after which the absorbance of theremaining solutions is measured. The standard curve used for amylosequantitation is of the form:Amylose (in micrograms)={(Absorbance of iodine solution at 635nm)−[(Slope of amylopectin standard curve in micrograms⁻¹)×(TotalCarbohydrate of Solution in micrograms)]}/[(Slope of amylose standardcurve in micrograms⁻¹)−(Slope of amylopectin standard curve inmicrograms⁻¹)].

The apparent amylose in micrograms is converted to a percentage of thetotal starch by dividing the value obtained by the carbohydrate contentof the solution and then multiplying by 100. Three independent analysesfor each starch were conducted. The results are presented in Table 7.

For analysis of the amylose content by gel permeation chromatography,samples consisted of samples of normal starch (lab isolated and acommercial normal starch from Cerestar-USA, C*Gel 03420), waxy starch(lab isolated and a commercial waxy starch from Cerestar-USA, C*Gel04230), and lab-isolated waxy-E starches. Starch granules (5.5 mg) in amicrocentrifuge tube are dispersed in 0.4 mL of 90% dimethyl sulfoxideby heating in a boiling water bath for one hour. The sample is agitatedevery 10 minutes during heating. The dispersed starch is precipitated byadding 1.6 mL of ethanol and centrifuged at 3000 g for five minutes in amicrocentrifuge at room temperature. The supernatant material isdiscarded. The starch pellet is washed twice with 1.0 mL of ethanol andonce with 1.0 mL of acetone, centrifuging the sample as described aboveeach time. The resultant non-granular starch is allowed to dry in theuncapped microcentrifuge tube for at least two hours. The drynon-granular starch is mixed with 0.9 mL of water and 0.1 mL of 100 mMsodium acetate (pH 4.5) and heated in a boiling water bath for one hour.The sample is mixed every 10 minutes during boiling. After heating, thesample is cooled to 40° C. in a water bath. An isoamylase suspension(0.001 mL; isolated from Pseudomonas sp., Megazyme International IrelandLtd, Co. Wicklow, Ireland) is added and the sample is inverted severaltimes before it is returned to the 40° C. water bath. After 18 hours,the sample is heated in a boiling water bath for five minutes toinactivate the enzyme. The sample is allowed to cool after which time0.2 mL of digest is added to 1.8 mL of DMSO. For injection, 0.5 mL iscentrifuged at 9,000×g. High-performance GPC is conducted in conjunctionwith a differential refractive index detector as part of achromatography system (Waters Breeze HPLC system consisting of a 1515Isocratic HPLC Pump. A Waters 2414 Differential Refractive Indexdetector, and a Rheodyne model 7725i injector with a 0.250 mL injectionloop, Waters Corporation, Milford, Mass.). Three PL-Gel 10 micrometerMixed-B (300×7.5 mm) analytical columns (Polymer Laboratories, Amherst,Mass.) and one PL-Gel 10 micrometer Mixed-B (100×7.5 mm) guard column(Polymer Laboratories, Amherst, Mass.) are used to separate thecomponent chains of the starch. The system is operated at a flow rate of0.5 mL/min with a mobile phase of 0.5% lithium bromide in DMSO. Thesystem is calibrated with standards of maltose (Sigma-Aldrich, St.Louis, Mo.), maltotriose (Sigma-Aldrich, St. Louis, Mo.), maltoheptaose(Sigma-Aldrich, St. Louise, Mo.), and pullulan standards with weightaverage molecular weights of 788000, 212000, 47300, 22800, 11800, and5900 from a carbohydrate standards kit (Polymer Laboratories, Amherst,Mass.). All injections are 0.200 mL. Monitoring of the chromatograms andanalysis of the data is done using the accompanying software (Breeze v.3.20). Chromatograms of EX68wx and EX68 normal starch are shown in FIG.2. Chromatograms of EX68wx, EX385wx-E1, and EX12wx-E2 are shown in FIG.3. From FIGS. 2 and 3 it is clear that negligible area is observedbefore 43 minutes for waxy starch. For normal starch, a minimum in thechromatogram is observed at 43 minutes; this minimum was used as ademarcation between the elution of amylose and debranched amylopectinfrom the system: amylose elutes before 43 minutes and debranchedamylopectin elutes after 43 minutes. The percentage of the area elutingbefore a time of 43 minutes in relation to the total area is calculatedand is used as a measure of the amylose content (w/w) of the starch. Incases where small areas were observed before 43 minutes, the time slicesof multiple chromatograms were averaged and the relative areas precedingand following 43.0 minutes for the average chromatogram were thencalculated. Amylose contents calculated in this way from allchromatograms are presented in Table 7.

From the GPC chromatograms, the AP Ratio (Amylopectin Chain Ratio) wascalculated. The AP Ratio is the ratio of the area of the chromatogrambetween a time of 46.01667 minutes and 51 minutes to the area of thechromatogram between a time of 43.01667 minutes and 46 minutes. TABLE 7Amylose Content of Starches Amylose Content High- AP Ratio PerformanceHigh- Spectrophotometric GPC^(a) Performance Sample (% w/w) (% w/w)GPC^(a) Normal EX68 Normal 23.3 ± 1.5  27.0 (1)  3.5 (1) C* Normal 20.0± 0.7  287.7 (1)  3.4 (1) wx starch EX68wx 0.4 ± 0.1 0.2 (3) 3.7 (3) C*waxy 1.7 ± 0.3 0.2 (2) 3.7 (2) wxae starch EX52wxae 17.3 ± 1.0  3.1 (2)1.3 (2) waxy-E starch EX56wx-E1 1.4 ± 0.2 1.3 (3) 3.6 (3) EX385wx-E1 2.4± 0.1 2.2 (3) 3.7 (3) EX78wx-E1 2.5 ± 0.2 2.3 (3) 3.7 (3) EX12wx-E2 6.5± 0.4 7.2 (1) 3.7 (1)^(a)The number of injections utilized for the calculation of the amylosecontent of the starch is shown in parentheses.

The results show that the amylose of EX56wx-E1, EX385wx-E1, EX78wx-E1,and EX12wx-E2 starches is significantly less than normal starch.Additionally, the results of these tests clearly show that the lowamylose starches have an anylose content which is also higher than thepure lab-isolated waxy starches. The higher amylose content of the C*waxy starch compared to the lab-isolated waxy starches is likely to bean artifact of normal starch contamination from commercial isolationprocesses. Additionally, the waxy-E starches may be divided into twogroups based on amylose content: one group which has an amylose contentbetween 1% and 3% (wx-E1 starches; EX56wx-E1, EX385wx-E1, EX78wx-E1) andanother group which has an amylose content between 6% and 8% (wx-E2starches; EX12wx-E2). Further, notice that for the EX52wxae starch theamylose contents determined by the two methods differ considerably. Thisis because wxae starches have an altered amylopectin structure which isable to produce some blue color resulting in highspectrophotometrically-determined amylose contents. However, when thesame starch is analyzed by chromatography, a very low proportion of thetotal area elutes from the chromatograph before a time of 43 min, andthis area is actually part of a peak which is attributable to theamylopectin of this starch (FIG. 2). Thus, there is no true amylose inthe wxae starch. For the low amylose starches, the distribution ofstarch chains eluting after 43 minutes is indistinguishable from waxystarch; long amylopectin chains are not responsible for the observedamylose of waxy-E starches. Thus the amylose of the waxy-E starches maybe quantitated both by spectrophotometric and chromatographicmeasurement techniques, and both techniques yield similar amylosecontent values. Additionally, to the best of our knowledge the lowamylose contents of the waxy-E starches are not attributable to longchain amylopectin. Further, the results show that the AP Ratio is within0.5 of the AP ratio of the normal starches. The wxae starch has an APRatio lower than Y2 that of the normal, waxy, and waxy-E starches,indicating the severe effects that the ae mutation has on the chaindistribution of amylopectin.

Example 4 Starch Gelatinization—Pasting Viscosity Profiles at pH 6.5-2.5min at 95° C.—waxy-E, waxy, and Normal Starches

This experiment was conducted to demonstrate that the waxy-E starcheshave unique gelatinization behavior. Assessment of the viscosity changesduring starch gelatinization are commonly conducted using a Rapid ViscoAnalyzer.

A pH 6.5 buffer solution is prepared as described in the “ApplicationsManual for the Rapid Visco Analyzer” (Anonymous. 1998. Ch. 7, Generalapplications, in the Applications Manual for the Rapid Visco Analyzer,Newport Scientific Pty. Ltd., Warriewood NSW, Australia, p. 20). Bothp-hydroxybenzoic acid methyl ester (0.8 g; Sigma-Aldrich, St. Louis,Mo.) and n-propyl p-hydroxybenzoate (0.2 g; Sigma-Aldrich) are added toa 250 mL beaker, to which 150 mL of water is added. The suspension isbrought to a boil with stirring to dissolve the solids. The hot solutionis added to 700 mL of distilled water in a 1000 mL graduated cylinder,after which the volume is brought to 1000 mL. To this solution is added:18.9 g of dibasic sodium phosphate heptahydrate (Fisher Scientific,Pittsburgh, Pa.), 2.0 g of sodium benzoate (Sigma-Aldrich), and 2.7 g ofanhydrous, granular citric acid (Sigma-Aldrich). The mixture is stirreduntil all of the solids are dissolved. Using a properly calibrated pHmeter, the mixture is then adjusted to pH 6.5 using citric acid if thepH is greater than 6.5 or dibasic sodium phosphate if the pH is below6.5. For each starch, a known mass of starch (on a dry weight basis) isweighed into an aluminum rapid visco analysis cup (Newport ScientificPty. Ltd). The sample is then brought to a total mass of 28 g with pH6.5 buffer. The RVA paddle is then added to the RVA cup and the paddlethen agitated in an up and down motion for 15 seconds to suspend thestarch. The cup, paddle, and starch suspension are then transferred tothe RVA and the instrument analysis procedure is immediately initiated.As the starch slurry is mixed at 960 rpm for the initial 10 seconds and160 rpm for the remainder of the RVA analysis while the temperature ismodulated using the controlling/analysis software (Thermocline forWindows v. 2.2, Newport Scientific Pty. Ltd.) per the following Standard1 Version 5 (December 1997) heating and stirring program: hold at 50° C.for one minute, heat to 95° C. over 3.7 minutes, hold at 95° C. for 2.5minutes, cool to 50° C. over 3.8 minutes, and hold at 50° C. for 2minutes. This method, when used with a Rapid Visco Analyzer 4 instrumentis the RVA Standard Method.

For this Example, 1.4 g of starch on a dry weight basis was used for alltests.

Analysis of this viscogram is conducted using the accompanying software.Data from three analyses of each starch are presented in Table 8,including samples of commercial starches obtained from Cerestar-USA(normal starch, C*Gel 03420; waxy starch C*Gel 02430). Exampleviscograms are presented in FIG. 4. TABLE 8 Rapid Visco Analysis ProfileData - Ph 6.5 - 2.5 min @ 95° C. Peak HP Break Final Peak PastingViscosity Viscosity Down B/P^(a) Viscosity Setback Time Temp Sample (cp)(cp) (cp) (%) (cp) (cp) (min) (° C.) Normal EX68  461 ± 13 435 ± 18 101± 4  21.9 428 ± 6  68 ± 6 6.1 ± 0.2 94.0 ± 1.6 C* Normal 305 ± 4 386 ±10 90 ± 4 29.6 333 ± 4  28 ± 3 6.6 ± 0.3 94.7 ± 0.5 wx starch EX68wx1133 ± 3  616 ± 4  573 ± 14 50.6 633 ± 10 73 ± 7 4.1 ± 0.0 75.5 ± 0.5EX56wx 1162 ± 18 635 ± 14 580 ± 3  49.9 646 ± 23 65 ± 6 4.1 ± 0.1 76.9 ±0.4 C* waxy 1293 ± 7  857 ± 24 521 ± 3  40.3 804 ± 3  33 ± 6 4.4 ± 0.177.5 ± 0.1 waxy-E starch EX56wx- 1149 ± 12 958 ± 16 324 ± 11 28.2 862 ±15 37 ± 4 5.7 ± 0.1 76.1 ± 0.4 E1 EX78wx- 1123 ± 10 1001 ± 10  269 ± 1024.0 956 ± 27 102 ± 22 6.1 ± 0.1 78.8 ± 0.6 E1 EX385wx- 1171 ± 22 1004 ±15  290 ± 21 24.8 1013 ± 13  132 ± 7  5.8 ± 0.2 77.5 ± 0.1 E1 EX12wx- 690 ± 28 710 ± 23 −121 ± 5  17.5 906 ± 24 337 ± 9  7.0^(b) 88.8 ± 1.1E1^(a)Percentage breakdown relative to the peak viscosity. B/P ={[Breakdown (cp)]/[Peak Viscosity (cp)]} × 100^(b)Peak time exceeded 7.0 minutes.

All of the low amylose starches and waxy-E starches had a significantlyhigher peak viscosity and higher final viscosity than the normalstarches, indicating that all of the waxy and waxy-E starches excel inthe development of viscosity at relatively low starch concentrations.Additionally, all waxy-E and waxy starches had a pasting temperaturelower than that of the normal starches, indicating that all of thesestarches begin to build viscosity earlier than do the normal starches.

The waxy-E starches differed from the waxy starches in many respects(Table 8). All of the low amylose starches had a breakdown viscosityless than waxy starches; this is true when viewed as the absolutebreakdown viscosity of the waxy-E starches but is also lower when thebreakdown viscosity is viewed as a percentage of the peak viscosity(B/P, Table 8) of the waxy-E starch. Additionally, all of the waxy-Estarches had a peak time later than those of the waxy starches. Thewaxy-E starches all had a final viscosity higher than those of the waxystarches. All of these observations indicate that the waxy-E starchesdevelop viscosity more slowly than do waxy starches and also retain andcontinue to develop viscosity during processing over a longer period oftime than do the waxy starches.

Further, the waxy-E starches may be divided into two groups of differingbehavior: one group containing EX56wx-E1, EX78wx-E1, and EX385wx-E1(wx-E1 Group, based on functional properties, see Table 8) and the othergroup containing EX12wx-E2 (wx-E2 Group, also based on functionalproperties, see Table 8). These groupings are the same as thosedescribed for the amylose content of these starches (See Example 3).Despite the common differences between all of these waxy-E starches andwaxy starches noted above, the process by which each group obtains theseproperties happens in a different way. Starches of the wx-E1 Groupdevelop a peak viscosity similar to that of the waxy starches, breakdown less than the waxy starches, and then set back an amount similar towaxy starches to result in a final viscosity higher than waxy starch.Starches of the wx-E2 Group plateau at a viscosity between the normalstarches and waxy starches without noticeable breakdown and then developconsiderable setback viscosity to result in a final viscosity higherthan waxy starch.

Example 5 Starch Gelatinization—Pasting Viscosity Profiles at pH 6.5-20min at 95° C.—waxy-E, waxy, and Normal Starches

This experiment was conducted to further demonstrate that the waxy-Estarches have unique gelatinization behavior. Assessment of theviscosity changes during starch gelatinization are commonly conductedusing a Rapid Visco Analyzer.

A pH 6.5 buffer solution is prepared as described in the “ApplicationsManual for the Rapid Visco Analyzer” (Anonymous. 1998. Ch. 7, Generalapplications, in the Applications Manual for the Rapid Visco Analyzer,Newport Scientific Pty. Ltd., Warriewood NSW, Australia, p. 20). Bothp-hydroxybenzoic acid methyl ester (0.8 g; Sigma-Aldrich, St. Louis,Mo.) and n-propyl p-hydroxybenzoate (0.2 g; Sigma-Aldrich) are added toa 250 mL beaker, to which 150 mL of water is added. The suspension isbrought to a boil with stirring to dissolve the solids. The hot solutionis added to 700 mL of distilled water in a 1000 mL graduated cylinder,after which the volume is brought to 1000 mL. To this solution is added:18.9 g of dibasic sodium phosphate heptahydrate (Fisher Scientific,Pittsburgh, Pa.), 2.0 g of sodium benzoate (Sigma-Aldrich), and 2.7 g ofanhydrous, granular citric acid (Sigma-Aldrich). The mixture is stirreduntil all of the solids are dissolved. Using a properly calibrated pHmeter, the mixture is then adjusted to pH 6.5 using citric acid if thepH is greater than 6.5 or dibasic sodium phosphate if the pH is below6.5. For each starch, 1.4 g of starch (dry weight basis) is weighed intoan aluminum rapid visco analysis cup (Newport Scientific Pty. Ltd). Thesample is then brought to a total mass of 28 g with pH 6.5 buffer. Asthe starch slurry is mixed at 960 rpm for the initial 10 seconds and 160rpm for the remainder of the RVA analysis while the temperature ismodulated using the controlling/analysis software (Thermocline forWindows v. 2.2, Newport Scientific Pty. Ltd.) per the following ST-01Revision 3 (November, 1998) heating and stirring program (NewportScientific Pty. Ltd.): hold at 50° C. for 0.5 minute, heat to 95° C.over 2.5 minutes, hold at 95° C. for 20 minutes, cool to 50° C. over 3.0minutes, and hold at 50° C. for 9 minutes. Analysis of the viscogram isconducted using the accompanying software. Data from three analyses ofeach starch are presented in Table 9, including samples of commercialstarches obtained from Cerestar-USA (normal starch, C*Gel 03420; waxystarch C*Gel 02430). Example viscograms are presented in FIG. 5.

All of the waxy-E starches and waxy starches had a significantly higherpeak viscosity and higher final viscosity than the normal starches,indicating that all of the waxy and waxy-E starches excel in thedevelopment of viscosity at relatively low starch concentrations.Additionally, all waxy-E and waxy starches had a pasting temperaturelower than that of the normal starches, indicating that all of thesestarches begin to build viscosity earlier than do the normal starches.

The waxy-E starches differed from the waxy starches in many respects(Table 9). All of the waxy-E starches had a peak time later than thoseof the waxy starches, indicating that the waxy-E starches developviscosity more slowly than do waxy starches and also retain and continueto develop viscosity under more severe temperature conditions than dothe waxy starches. Additionally, the waxy-E starches developsignificantly higher setback viscosities and final viscosities than dowaxy starches, attributable to the structure-developing amylose in thewaxy-E starches.

Further, as in Examples 3 and 4, the waxy-E starches may be divided intotwo groups of differing behavior: one group containing EX56wx-E1,EX78wx-E1, and EX385wx-E1 (wx-E1 Group, based on functional properties,see Table 9) and the other group containing EX12wx-E2 (wx-E2 Group, alsobased on functional properties, see Table 9). Despite the commondifferences between all of these waxy-E starches and waxy starches notedabove, the process by which each group obtains these properties happensin a different way. Starches of the wx-E1 Group develop a peak viscositysimilar to that of the waxy starches, and then set back to result in afinal viscosity higher than waxy starch. Starches of the wx-E2 Groupplateau at a viscosity between the normal starches and waxy starches andthen develop considerable setback viscosity to result in a finalviscosity higher than waxy starches. TABLE 9 Rapid Visco AnalysisProfile Data - Ph 6.5 - 2.5 min @ 95° C. Peak HP Break Final PeakPasting Viscosity Viscosity Down B/P^(a) Viscosity Setback Time TempSample (cp) (cp) (cp) (%) (cp) (cp) (min) (° C.) Normal EX68 483 ± 3 427± 3  76 ± 8 15.7 609 ± 39 202 ± 48 4.4 ± 0.0 95^(b) C* Normal 409 ± 6378 ± 6   89 ± 24 21.8 408 ± 50  88 ± 27 4.7 ± 0.1 95^(b) wx starchEX68wx 1096 ± 22 531 ± 10 761 ± 17 69.4 509 ± 10 174 ± 5  2.9 ± 0.1 77.2± 0.4 EX56wx 1160 ± 27 561 ± 3  788 ± 19 67.9 532 ± 22 160 ± 17 3.0 ±0.0 77.7 ± 0.3 C* waxy 1212 ± 37 866 ± 42 709 ± 35 58.5 624 ± 18 122 ±20 3.3 ± 0.0 78.7 ± 0.6 waxy-E starch EX56wx-E1 1205 ± 23 889 ± 31 747 ±21 62.0 693 ± 27 235 ± 24 4.4 ± 0.1 77.2 ± 0.3 EX78wx-E1 1171 ± 13 933 ±6  668 ± 24 57.0 698 ± 13 196 ± 24 5.0 ± 0.1 79.7 ± 0.5 EX385wx-E1 1194± 7  911 ± 15 684 ± 16 57.2 865 ± 32 355 ± 13 4.6 ± 0.0 78.6 ± 0.5EX12wx-E1  783 ± 12 786 ± 13 243 ± 4  31.0 755 ± 12 216 ± 8  6.9 ± 0.188.3 ± 4.1^(a)Percentage breakdown relative to the peak viscosity. B/P ={[Breakdown (cp)]/[Peak Viscosity (cp)]} × 100^(b)Peak time exceeded 7.0 minutes.

Example 6 Starch Gelantinization—Pasting Viscosity Profile at pH 6.5-2.5min at 95° C.—Mixtures of waxy and Normal Starch

This experiment was conducted to demonstrate that the properties ofwaxy-E starches cannot be reproduced using mixtures of normal starch andwaxy starch.

Mixtures of normal starch and waxy starch were prepared to producestarches with bulk amylose content within the range observed for thewaxy-E starches. The starches examined were the waxy and normal starchesof EX68 used in Example 4. The composition of the mixtures prepared andthe amylose content of the mixtures is illustrated in Table 10. Theamylose content of the normal starch was assumed to be 20% for thisexperiment. TABLE 10 Composition of starch mixtures Estimated AmyloseContent Normal Starch EX68 Waxy Starch EX68wx (%) (dry mass %) (dry mass%) 0 0 100 2 10 90 4 20 80 6 30 70 8 40 60

The starch pastes were prepared using the RVA Standard Method. For thisExample, 1.4 g of total starch on a dry weight basis was used for alltests. The properties of the mixtures are shown in Table 11.

Addition of the normal starch to the waxy starch had four clear effectson the bulk properties of the starch: (1) the peak viscosity decreasedwith increasing normal starch content, (2) the breakdown of the starchdecreased with increasing normal starch content as indicated by both theabsolute value of the breakdown and by the B/P ratio, (3) the setbackviscosity of the starch increased with the inclusion of normal starch,and (4) the peak time of the starch increased with increasing starchcontent. Some of these behaviors appear to mimic those of the waxy-Estarches, especially those of the wxE1 Group (Example 4), however:

1) for the wx-E1 Group waxy-E starches higher peak viscosities areobserved compared to the 80% EX68wx/20% EX68 mixture which shows aconsiderable drop in peak viscosity compared to the 100% EX68wx starch.

2) The wx-E1 Group waxy-E starches retain considerably more viscosity asa hot paste and at their minimum viscosities compared to the 80%EX68wx/20% EX68 mixture which has hot paste and minimum viscositiessimilar to or lower than those of the EX68wx starch. The decreasing B/Pratio for the mixtures of waxy and normal starch with increasing normalstarch appears to be primarily due to the decreasing peak viscosities ofthe mixtures with increasing amylose normal starch content rather than adecrease in breakdown viscosity. TABLE 11 Rapid Visco Analysis ProfileData - Ph 6.5 Peak HP Final Peak Pasting Viscosity Viscosity BreakdownB/P^(a) Viscosity Setback Time Temp Sample (cp) (cp) (cp) (%) (cp) (cp)(min) (° C.) EX68wx 1133 ± 3 616 ± 4 573 ± 14 50.6 633 ± 10 73 ± 7 4.1 ±0.0 75.5 ± 0.5 90% 1065 585 533 50.0 631 99 4.2 75.9 EX68wx  2% amylose80% 1029 619 472 45.9 677 120 4.3 76.0 EX68wx  4% amylose 70% 971 614411 42.3 682 122 4.4 75.9 EX68wx  6% amylose 60% 903 628 333 36.9 675105 4.8 75.9 EX68wx  8% amylose^(a)Percentage breakdown relative to the peak viscosity. B/P ={[Breakdown (cp)]/[Peak Viscosity (cp)]} × 100

The wx-E1 Group waxy-E starches have higher pasting temperatures andpeak times than the mixtures of waxy and normal starch; these propertiesappear to be dominated by the waxy content of the mixtures. All of thesepoints indicate that the waxy-E starch properties cannot be reproducedby blending of normal starch with waxy starch.

Example 7 Starch Paste Texture—Rheology

This experiment was conducted to demonstrate the rheological propertiesof waxy-E starch.

Starch pastes were prepared with waxy starches (EX68wx, EX56wx,Cerestar-USA commercial waxy starch C*Gel 02430) and waxy-E starches(Ex385wx-E1, EX78wx-E1, EX56wx-E1, and EX12wx-E1). Normal starchesgelled during storage in preliminary experiments, an indication of theirinstability and high elastic modulus, so they could not testedTheologically.

Starches were cooked using the RVA Standard Method. For this Example,1.4 g of starch on a dry weight basis was used for all tests.Immediately after cooking, each paste was transferred to a 50 mL tubeand placed in a 25° C. water bath. Samples were analyzed using arheometer 18-22 hours later. After storage, frequency and straindependence of the starch pastes were tested using a rheometer (RFSIIIFluids Spectrometer, Rheometric Scientific, Piscataway N.J.). All pasteswere measured at 25° C. A parallel plate geometry was utilized fortesting (50 mm; 0.9 to 1.1 mm gap width); loaded samples were permittedto rest between the plates of the rheometer for 10 minutes in order toreduce the effects of loading on the measurements. A thin film of oilwas applied to the exposed surface of the paste between the rheometerplates to minimize moisture evaporation during the testing process.Frequency dependence of a paste was always examined first, followed bythe strain dependence. The frequency dependence of the pastes was testedbetween 0.1 and 100 radians per second with a oscillatory strain of 1%.Strain dependence of the pastes was tested between 0.1 and 1000%deformation at a constant testing frequency of 1 radian per second. TheEM of a starch is the elastic modulus of the starch below the yieldstrain and at an oscillatory frequency of 1 rad/sec as observed usingthis testing method after the starch has been cooked using the RVAStandard Method using a concentration of starch such that the finalviscosity of a waxy starch extracted from a plant of the same species isbetween 600 and 850 centipoise and after the cooked starch has beenstored for 18-22 hours at 25° C.

Two replicates of the experiment were conducted, and the analysis orderof the second replicate was the reverse of the first replicate in anattempt to eliminate any confounding effect of storage time on theresults. The strain and frequency dependence of the starch pastes ispresented in Table 12. The results of each replicate are shown.Illustrative charts of G′ and phase angle vs frequency and G′ and phaseangle vs strain are presented in FIGS. 6 and 7, respectively. The waxy-Estarch pastes had a lower frequency dependence than did any of the waxystarch pastes (Table 12), with approximately a two fold increase in G′between a frequency of 0.1 and 100 radians per second compared to waxystarch pastes which generally had a 5 fold increase over the samefrequency range. The lower frequency dependence of waxy-E starch pastesshows that the waxy-E starch pastes have more gel-like character than dowaxy starch pastes. TABLE 12 Rheology of Starch Pastes - Storage at 25°C. Frequency Dependence Strain Dependence 0.1 rad/s 100 rad/s 1% 200%1000% G′ phase angle G′ phase angle G′ phase angle G′ phase angle G′phase angle Starch Source (Pa) (deg) (Pa) (deg) (Pa) (deg) (Pa) (deg)(Pa) (deg) wx starch EX68wx 2 22 12 37 3 25 2 36 1 52 2 18 13 37 3 24 237 1 50 EX56wx 2 19 13 36 3 22 2 32 1 49 2 17 13 36 3 23 2 36 1 50 C*waxy 3 14 15 33 5 15 3 22 2 39 3 12 16 33 5 17 3 24 2 40 waxy-E starchEX56wx-E1 27 4 49 16 32 4 21 16 3 63 26 4 46 17 29 5 21 16 4 63EX385wx-E1 41 3 66 12 46 3 26 23 3 68 41 3 65 13 44 4 26 24 3 69EX78wx-E1 24 6 51 17 29 6 16 20 2 64 22 6 49 18 26 7 17 19 3 64EX12wx-E2 39 5 79 16 43 6 10 42 1 74 40 5 81 16 46 6 11 41 1 74

The waxy-E starch pastes had a higher elastic modulus at 1% strain thanthe elastic modulus of waxy starch pastes, exceeding nearly 10 fold inall cases. Additionally, the phase angles of waxy-E starch pastes at 1%strain were lower compared with the phase angles of waxy starch pastes,indicating that a higher proportion of the complex modulus of waxy-Estarch pastes is attributable to the elastic component of the pastecompared to waxy starch pastes. Thus, the waxy-E starch pastes areconsiderably different Theologically from waxy starch pastes.

The elastic modulus of waxy-E starch pastes remained higher than theelastic modulus of waxy starch pastes through 500-1000% strain.Additionally, through 100-200% strain waxy-E starch pastes generallymaintained lower phase angles than waxy starch pastes. Thus, waxy-Estarch pastes not only retained a relatively high elastic modulus butalso a relatively high elasticity (as a component of the complexmodulus, indicated by the low phase angles) through high deformationscompared to waxy starch pastes.

This combination of a moderate elastic modulus and low phase angleindicates that under low deformations the waxy-E starches behave morelike gels than viscous pastes through 1% to 200% strain. Further, thewaxy-E starches may be divided into two groups (wx-E1 and wx-E2) as inthe previous examples, with the starch of the wx-E2 group yielding at alower strain than the wx-E1 starches. Regarding all of the waxy-Estarches, their gel behavior is unusual for a native starch: waxystarches, as Table 12 shows, do not develop a high elastic modulus andhave a high phase angle even at low strains, and gels of normal starchor amylose are sensitive to small deformations (see additionally Example8), often losing considerable elastic modulus between 0.1% and 1% strainlike other strong biopolymer gels.

Example 8 Starch Paste Texture—Penetrometry

This experiment was conducted to demonstrate that waxy-E starches havethe ability to develop gels, unlike waxy starches at the sameconcentration, and that the gels of waxy-E starches do not have the sameproperties as normal starch gels. Penetrometry is conducted using amethod modified from Takahashi and Seib (1988, Cereal chemistry65:474483) and Yamin et al (1999, Cereal Chemistry 76:175-181). Well inadvance of the experiment, a cylindrical plastic sample receptacle (58mm tall by 22 mm inside diameter) with a screw-on lid is prepared bysawing it along its long axis. The receptacle halves are welded togetherwith silicone adhesive, taking care to match the threads for thescrew-on lid at the open end of the receptacle. The adhesive holding thewelded receptacle together is then permitted to dry for 48 hours. Starchis pasted as a 10% (w/w) slurry in pH 6.5 phosphate buffer in the RapidVisco Analyzer (Rapid Visco Analyser 4, Newport Scientific Pty. Ltd.):while stirring at 160 rpm, the sample is held at 50° C. for one minute,heated to 95° C. over 3.7 minutes, held at 95° C. for 2.5 minutes,cooled to 50° C. over 3.8 minutes, and then held at 50° C. for 2minutes. Upon immediate completion of sample preparation using the RVA,the sample receptacle is filled by the resultant gelatinized paste fromthe RVA. The full receptacle is then covered with its screw-on cap andthen the cap and a portion of the sample receptacle is wrapped withlaboratory film. The gelatinized starch paste is stored for seven daysat 4° C. Before analysis, a starch sample is removed from refrigeratedstorage, and allowed to equilibrate to room temperature for two hours.For analysis, the halves of the sample receptacle are separated and thegel (when present) is cut along its short axis into two pieces: doing soprovides two samples for analysis with no edge effects due to the samplebeing in contact with either the bottom or top of the sample receptacle.These “contact” edges are trimmed as necessary to provide a horizontalsurface for gel testing. The prepared gels are analyzed using apenetrometer (Texture Analyzer TA-XT2i with a 5 kg load cell, StableMicro Systems, England) interfaced with a computer running associateddata analysis and instrument control software (Texture Expert Exceedversion 2.55, Stable Micro Systems, England). Analysis was done using amethod modified from Yamin et al (1999, Cereal Chemistry 76:175-181). Agel sample 1.5 cm in height is penetrated with a cylindrical probe witha flat surface having a diameter of 4 mm. The gel is compressed 7.5 mmat a rate of 0.9 mm/s by the probe and withdrawn at the same rate. Thepeak force observed during penetration of the gel was the hardness. Thefracturability was the initial force peak observed during penetration ofthe sample, related to the initial penetration of the sample by thedownward-moving probe. The force during penetration as the area of thecurve (in gram-seconds) was the gel firmness. The positive force as thearea of the curve (in gram-seconds) acting on the probe during itswithdrawal was recorded. The resilience (or resiliency) of the gel wascalculated as the ratio of the positive force during probe withdrawal tothe positive force during probe penetration (the firmness). Tenmeasurements per gel were conducted and the two highest and two lowestmeasurements of each property were removed; these data were typicallybeyond two standard deviations of the mean. Penetrometry data, as theaverage of six penetrations per gel, are presented in Table 13. Theexperiment was conducted in duplicate; results from both gels preparedfor each starch are presented in Table 13.

The results of these tests clearly show that waxy-E starches can developa range of textures which is not developed by either waxy-E or normalstarch. All of the waxy-E starches have a hardness and firmness belowthat of normal starches. Additionally, the quality of waxy-E starch gelsis not similar to those formed by normal starch: waxy-E starch gels donot fracture as do normal starch gels. Instead, the waxy-E gels whichare formed are highly resilient and deformable with a gradual increasein force during penetration and a gradual release of that force duringremoval of the probe from the starch gel. This behavior is consistentwith the high deformability of waxy-E starch pastes observed usingdynamic oscillatory rheometry (Example 7). TABLE 13 Starch Gel TextureProperties Probe Withdrawal Positive Hardness Firmness Area ResilienceSample Facturability (g) (g-s) (g-s) (%) Normal EX68 30 ± 1   V^(a) 145± 9 17 ± 3 12 31 ± 1 V 177 ± 4 19 ± 1 11 C* Normal 35 ± 1 V 191 ± 3 25 ±2 13 34 ± 2 V 213 ± 6 21 ± 3 10 wx starch EX68wx  NA^(b) NA NA NA NA NANA NA NA NA C* waxy NA NA NA NA NA NA NA NA NA NA waxy-E starchEX56wx-E1   ND^(c) 2.3 ± 0.2  5.4 ± 0.6  4.4 ± 0.6 81 ND 2.9 ± 0.2  8 ±1  6.4 ± 0.9 80 EX78wx-E1 ND 5.4 ± 0.2  14.0 ± 0.9 11.3 ± 0.8 80 ND 6.7± 1.1   20 ± 3.8 12.2 ± 0.9 61 EX385wx-E1 ND 6.6 ± 0.7  21 ± 3 16 ± 2 76ND 6.0 ± 0.3  18 ± 2 15 ± 1 83 Ex12wx-E2 ND 4.6 ± 0.4  13 ± 2  8.1 ± 0.962 ND 4.0 ± 0.3  11 ± 2  6.8 ± 0.7 62^(a)V = variable Hardness varied considerably after the initial fractureof the gel for these samples. Results are not reported but weregenerally of the same magnitude as the hardness.^(b)NA = not applicable. Starch was a viscous sol which could not bemeasured.^(c)ND-not detected. No initial fracture point was observed for thesegels. Instead, the force continued to steadily increase until themaximum penetration depth was reached.

Example 9 Starch Gelatinization—Calorimetry

This experiment was conducted to illustrate the temperature range andgranule stability of waxy-E starches. As described earlier, assessmentof the gelatinization temperature profile of the starch is commonlyconducted using a differential scanning calorimeter (DSC).

For each starch, 8.0 mg (±0.2 mg) of starch (dry weight basis) isweighed into a 0.05 mL stainless steel DSC sample pan. The sample isthen brought to a total mass of approximately 30 mg with water,resulting in a suspension of 25% starch (w/w). The mass of starch andwater is recorded and the starch concentration calculated based n themass of water added and the solids content of the starch. The pan isthen sealed and stored at room temperature for approximately 18 hours.The sample is heated in the DSC (Pyris 1 Differenetial ScanningCalorimeter, PerkinElmer Instruments, Norwalk, Conn.) from 5° C. to 140°C. at 10° C./min. The onset temperature, peak temperature, endsettemperature, and enthalpy of the gelatinization and amylose-lipidcomplex (if observed) endotherms are calculated using thecontrolling/analysis software (Pyris Software v. 3.81, PerkinElmerInstruments). Amylose-lipid complex enthalpy is determined as a partialarea of the total endotherm after 85° C. for wild type starch whenoverlap is observed between the starch gelatinization and amylose-lipidcomplex dissociation endotherms when necessary. An empty stainless steelpan is used as a reference and temperature and enthalpy calibrations aremade using an indium standard. Gelatinization data as averages of atleast three replicates are presented in Table 14. TABLE 14 StarchGelatinization Temperatures and Enthalpy Onset Peak Endset Temp TempTemp Enthalpy Sample (° C.) (° C.) (° C.) (J/g) Normal Ex68 Starch- 68.4± 0.2 71.9 ± 0.1 76.7 ± 0.2 16.8 ± 0.5 Starch AM-Lipid NA* NA* NA*  2.9± 0.5 C* Normal Starch- 69.8 ± 0.3 73.1 ± 0.2 77.9 ± 0.2 17.3 ± 0.4Starch AM-Lipid 83.9 ± 1.6 98.8 ± 2.9 107.4 ± 0.6   2.2 ± 0.3 wx starchEX68wx 67.8 ± 0.1 74.0 ± 0.3 79.3 ± 0.2 18.7 ± 0.4 EX56wx 67.4 ± 0.071.0 ± 0.0 76.1 ± 0.1 17.8 ± 0.5 C* waxy 66.8 ± 0.3 74.0 ± 0.1 79.5 ±0.3 18.8 ± 0.4 wxae starch EX52wxae 75.1 ± 0.4 85.7 ± 0.4 95.0 ± 0.623.5 ± 0.4 waxy-E starch EX56wx-E1 67.5 ± 0.4 70.9 ± 0.2 76.3 ± 0.5 18.8± 0.8 EX385wx-E1 66.1 ± 0.1 72.1 ± 0.3 79.1 ± 0.1 18.3 ± 0.8 EX78wx-E168.8 ± 0.1 74.3 ± 0.1 79.5 ± 0.4 18.6 ± 0.3 EX12wx-E2 Starch- 70.3 ± 0.274.3 ± 0.3 80.5 ± 0.1 19.5 ± 0.7 Starch AM-Lipid 87.8 ± 2.0 101.0 ± 0.2 107.1 ± 1.5   1.1 ± 0.2*NA—Not Applicable.Enthalpy was determined as a partial area of the total.Onset Temp, Peak Temp, and Endset Temp were not observed for thisendotherm.

The results of these tests clearly show that the waxy-E starches aresimilar in both gelatinization temperature range and enthalpy to waxystarches and normal starch. For at least one waxy-E starch, sufficientamylose-lipid complex enthalpy is also present for detection duringgelatinization using DSC. The lower amylose-lipid complex enthalpyobserved for waxy-E starches compared to normal starches is consistentwith the lower amylose content of waxy-E starches.

Example 10 Starch Stability—Calorimetry

This experiment was conducted to illustrate the paste stability ofwaxy-E starches. As described above, starch can reorganize aftergelatinization. The process of reorganization is called retrogradation.The amount of reorganization, an assessment of the temperature stabilityof starch, is commonly conducted using a differential scanningcalorimeter (DSC).

Samples examined for their gelatinization properties in the previousexample (above) were cooled to 5° C. and immediately placed in arefrigerator, where they were stored for seven days. After storage, thesamples were removed from the refrigerator, immediately placed in theDSC chamber at 5° C., and reheated in the DSC (Pyris 1 DifferentialScanning Calorimeter, PerkinElmer Instruments, Norwalk, Conn.) from 5°C. to 140° C. at 10° C./min. The onset temperature, peak temperature,endpoint temperature, and enthalpy of the retrogradation endotherm(s)are calculated using the controlling/analysis software (Pyris Softwarev. 3.81, PerkinElmer Instruments). An empty pan was used as a referenceand temperature and enthalpy calibrations were made using an indiumstandard. This method was used to determine the Retrogradation Enthalpyof the starch. Retrogradation data are presented in Table 15. TABLE 15Starch Retrogradation Temperatures and Enthalpy Onset Peak Endset TempTemp Temp Enthalpy Sample (° C.) (° C.) (° C.) (J/g) Normal EX68 Starch-35.6 ± 0.1 51.9 ± 0.6 65.1 ± 0.2 7.8 ± 0.2 Starch AM-Lipid 87.9 ± 0.897.5 ± 0.4 105.2 ± 0.8  1.6 ± 0.4 C* Normal Starch- 34.8 ± 0.9 51.3 ±0.9 65.3 ± 0.8 8.6 ± 0.2 Starch AM-Lipid 87.7 ± 0.4 96.5 ± 0.6 105.6 ±2.7  1.4 ± 0.1 wx starch EX68wx 40 ± 6 54.2 ± 2.1 66.3 ± 0.3 2.9 ± 0.3C* waxy 35.2 ± 1.0 57.0 ± 1.4 68.1 ± 4.9 2.8 ± 0.4 wxae starch EX52wxae36.2 ± 0.4 70.8 ± 0.5 84.5 ± 0.5 13.9 ± 0.6  waxy-E starch EX56wx-E134.4 ± 0.6 54.9 ± 0.5 64.8 ± 0.4 4.3 ± 0.4 EX385wx-E1 34.2 ± 0.4 55.8 ±0.6 65.8 ± 0.5 5.4 ± 0.6 EX78wx-E1 Starch- 34.8 ± 1.3 54.2 ± 1.0 65.5 ±0.8 6.7 ± 0.7 Starch AM-Lipid   93 ± 5.0 98 ± 2 108 ± 3  0.31 ± 0.07EX12wx-E2 Starch- 34.9 ± 1.1 53.2 ± 0.9 65.0 ± 0.6 7.8 ± 0.8 StarchAM-Lipid 90 ± 3 94.4 ± 0.2 106 ± 2  1.0 ± 0.2*NA = Not Applicable.The endotherm for this starch was bimodal, resulting in unreliableestimates of the onset temperature.

The waxy-E starches are between waxy starches and normal starches intheir retrogradation enthalpy, indicating that the waxy-E starches haveintermediate low temperature stability. All of the waxy-E starches havea retrogradation enthalpy lower than or equivalent to normal starches.For at least two waxy-E starches, sufficient amylose-lipid complexenthalpy is also present for detection during retrogradation analysisusing DSC. The lower amylose-lipid complex enthalpy observed for waxy-Estarches compared to normal starches is consistent with the loweramylose content of waxy-E starches.

Example 11 Starch Structure—High Performance Anion ExchangeChromatography

This test was conducted to illustrate the short chain distribution ofwaxy-E starches. Starch granules (5.5 mg) in a microcentrifuge tube aredispersed in 0.4 mL of 90% dimethyl sulfoxide by heating in a boilingwater bath for one hour. Samples consisted of two independent events ofwaxy starch from the EX68 line (EX68wx1 and EX68wx2), a wxae starch(EX52wxae), and four waxy-E starches (EX56wx-E1, EX385wx-E1, EX78wx-E1,and EX12wx-E1). The sample is agitated every 10 minutes during heating.The dispersed starch is precipitated by adding 1.6 mL of ethanol andcentrifuged at 3000×g for five minutes in a microcentrifuge at roomtemperature. The supernatant material is discarded. The starch pellet iswashed twice with 1.0 mL of ethanol and once with 1.0 mL of acetone,centrifuging the sample as described above each time. The resultantnon-granular starch is allowed to dry in the uncapped microcentrifugetube for at least tw hours. The dry non-granular starch is mixed with0.9 mL of water and 0.1 mL of 100 mM sodium acetate (pH 4.5) and heatedin a boiling water bath for one hour. The sample is mixed every 10minutes during boiling. After heating, the sample is cooled to 40° C. ina water bath. An isoamylase suspension (0.001 mL; isolated fromPseudomonas sp., Megazyme International Ireland Ltd, Co. Wicklow,Ireland) is added an the sample is inverted several times before it isreturned to the 40° C. water bath. After 18 hours, the sample is heatedin a boiling water bath for five minutes to inactivate the enzyme. Thesample is allowed to cool before 0.4 mL are centrifuged through a 0.22micron filter. This filtered sample is immediately injected into theHPAEC system. HPAEC is conducted in conjunction with a pulsedamperometric detector (PAD) as part of a chromatography system (DionexDX 500 chromatography system with a GP40 Gradient Pump, an ED40Electrochemical Detector, and a Rheodyne model 9125 injector with a0.050 mL injection loop, Dionex Corp, Sunnyvale, Calif.). A Carbopac PA1(4×250 mm) analytical column (Dionex Corp, Sunnyvale, Calif.) with aCarbopac PA1 (4×50 mm) guard column (Dionex Corp, Sunnyvale, Calif.) isused to separate the component chains of the starch. The system isoperated at a flow rate of 1.0 mL/min with a gradient profile of 150 mMsodium hydroxide (mobile phase “A”) and 500 mM sodium acetate in 150 mMsodium hydroxide (mobile phase “B”) as follows: 0 minutes, A:B::80:20; 2minutes, A:B::80:20; 10 minutes, A:B::70:30; 20 minutes, A:B::50:50; 60minutes, A:B::20:80; 80 minutes, A:B::20:80. All gradients in theprofile are linear. The system is calibrated with of a mixture ofglucose and oligosaccharides with a degree of polymerization (DP)between 2 and 7. Peaks appearing after DP 7 in the debranched starchchromatograms are presumed to have a DP a single glucose unit longerthan the previously eluting peak. All injections are 0.050 mL.Monitoring of the chromatograms and analysis of the data is done usingthe accompanying software (Peaknet v. 4.30). None of the starchescontained chains with a DP less than 6. A chromatogram of EX12wx-E2 isshown in FIG. 8. The percentage that each peak represents in relation tothe total peak area through a DP of 50 is calculated. These percentagesare presented in Table 16. Relative area percentage plots of EX68wx1,EX12wx-E2, and EX52wxae are illustrated in FIG. 9. TABLE 16 AreaPercentages of Debranched Non-Granular Starch through DP 50 EX68 EX68EX52 EX56 EX385 EX78 EX12 DP wx1 wx2 wxae wx-E1 wx-E1 wx-E1 wx-E2  60.621 0.805 0.432 0.718 0.760 0.862 0.621  7 0.801 1.054 0.609 0.9890.991 1.055 0.801  8 1.121 1.313 0.881 1.260 1.422 1.342 1.121  9 2.8342.830 1.576 3.016 2.885 2.992 2.834 10 4.426 4.142 2.310 4.600 4.1354.594 4.426 11 5.186 4.768 3.108 5.198 4.675 5.120 5.186 12 5.630 5.1323.815 5.625 5.104 5.602 5.630 13 5.845 5.402 4.272 5.826 5.261 5.7475.845 14 5.837 5.434 4.563 5.832 5.351 5.671 5.837 15 5.642 5.251 4.6055.654 5.254 5.525 5.642 16 5.369 4.975 4.622 5.396 4.957 5.275 5.369 174.923 4.457 4.361 4.880 4.591 4.862 4.923 18 4.488 4.095 4.188 4.4534.201 4.420 4.488 19 4.188 3.990 4.091 4.137 3.977 4.139 4.188 20 3.8983.787 4.095 3.914 3.722 3.856 3.898 21 3.620 3.545 3.850 3.554 3.5083.463 3.620 22 3.304 3.286 3.744 3.263 3.256 3.194 3.304 23 2.983 2.9943.437 3.069 2.977 2.690 2.983 24 2.628 2.671 3.219 2.290 2.718 2.2772.628 25 2.322 2.467 3.029 2.094 2.469 2.139 2.322 26 2.043 2.277 2.5981.901 2.248 1.975 2.043 27 1.828 2.051 2.507 1.669 2.032 1.682 1.828 281.548 1.845 2.208 1.479 1.825 1.552 1.548 29 1.359 1.647 1.940 1.3701.654 1.440 1.359 30 1.252 1.529 1.180 1.202 1.481 1.154 1.252 31 1.1331.335 1.678 1.115 1.343 1.038 1.133 32 1.014 1.246 1.518 0.928 1.2340.974 1.014 33 0.935 1.174 1.321 1.009 1.155 0.986 0.935 34 0.853 1.0051.433 0.905 1.059 0.930 0.853 35 0.830 0.974 1.056 0.819 1.015 0.8950.830 36 0.822 0.966 1.109 0.794 0.888 0.819 0.822 37 0.781 0.908 1.1160.813 0.909 0.929 0.781 38 0.809 0.915 1.052 0.787 0.816 0.899 0.809 390.809 0.903 1.135 0.829 0.879 0.788 0.809 40 0.786 0.855 1.092 0.7680.878 0.854 0.786 41 0.780 0.869 1.096 0.846 0.858 0.842 0.780 42 0.7630.813 1.067 0.764 0.910 0.940 0.763 43 0.745 0.864 1.098 0.846 0.8520.912 0.745 44 0.817 0.780 1.258 0.871 0.857 0.904 0.817 45 0.822 0.9021.214 0.846 0.861 0.745 0.822 46 0.732 0.716 1.081 0.789 0.854 0.9220.732 47 0.712 0.789 1.226 0.751 0.822 0.791 0.712 48 0.810 0.755 1.2840.783 0.790 0.753 0.810 49 0.697 0.735 1.146 0.752 0.804 0.784 0.697 500.656 0.747 1.148 0.597 0.763 0.666 0.656 SUM 100 100 100 100 100 100100

As a proportion of the chains with a DP of 50 or less, the chaindistribution of each waxy-E starch is not representative of the rangeobserved for wxae (EX52wxae) starch. Instead, the chain distributions ofthe waxy-E starches are representative of the chain distributionsobserved for the waxy starch. If long amylopectin chains were the causeof the cooking and physical properties and amylose content of the starchillustrated in Examples 3, 4, 5, 7, and 8, the chain distribution wouldbe expected to more closely resemble that of the wxae starch. Thischromatographic analysis is consistent with the appearance of the highperformance size exclusion chromatograms (FIG. 3) used to calculate theamylose content of the starches (Example 3).

Example 12 Determination of the Presence and Activity of StarchBiosynthetic Enzymes within Kernels

This set of experiments was conducted to demonstrate that the waxy-Estarch contains an active granule-bound starch synthase (GBSS) withreduced activity compared to normal starch and that commercial waxystarches and lab-isolated waxy starches lack such activity.

Starch Extraction and Protein Analysis of Starch Granules

The dry weight of a known number (1-5 kernels) from each sample wasrecorded and the kernels were ground initially using a retail coffeebean grinder. This was followed by homogenization using a Pro400homogenizer (Pro-Scientific Inc., Monroe, Conn., USA) in TEB (Tissueextraction buffer; 50 mM MES (pH 7.5), 1 mM EDTA, 5 mM DTT) at 4° C. Theresulting slurry was filtered through 4 to 6 layers of cheese-cloth andcentrifuged at 10,000 rpm for 10 minutes at 4° C. Supernates were savedand the pellets were washed 2× with TEB followed by a wash with 2%sodium dodecyl sulphate (SDS) solution. The pellet was again suspendedin TEB and microfuged at 10,000 rpm at 4° C. The supernate from thiswash was discarded and the pellets were stored at −80° C. until furtherused. Granular associated proteins were recovered by boiling starch for10 minutes in the presence of SDS-sample loading buffer (57 mM pH 6.8Tris-HCl, 2% SDS, 9% Glycerol, and a reducing agent plus bromophenolblue). The resulting slurry was cooled to room temperature andmicrofuged at 10,000 rpm for 10 minutes. The supernate with starchgranular proteins was retained for electrophoresis (below). Theseproteins were either run on SDS-PAGE or native-PAGE in order to detectthe protein levels and their activities, respectively (according to theprocedures described below).

SDS-PAGE (Denaturing and Non-Denaturing) and Detection of EnzymeActivity

Polyarcylamide (37.5:1 w/w acrylamide:bis-acrylamid) gels of either 8%straight (for Biorad MiniProtean III apparatus) or 7% to 20% gradient(Biorad Protean II) under non-denaturing conditions, and 10% or 12%under denaturing conditions (with 0.1% sodium dodecyl sulphate (SDS))were run according to Laemmli (Laemmli, U.K., 1970, Nature 227:680-685).The non-denaturing gels contained either 0.1% rabbit liver glycogen orpotato amylopectin and were electrophoresed in a running buffer (25 mMTris, 192 mM glycine, 1% SDS) containing 5 mM DTT. The denaturing gelscontained either 0.1% rabbit liver glycogen or potato amylopectin andwere electrophoresed in a running buffer without DTT. At the end of theelectrophoresis, denaturing gels were incubated in the renaturationbuffer (40 mM Tris, 5 mM DTT) for 90 minutes to 2 hours with a change ofsolution after every 30 minutes. The non-denaturing gels were incubatedin 5 to 10 ml of the reaction buffer [10 mg/ml glycogen, 5 mM ADPG, 5 mMglutathione, 0.5 mg/ml BSA, 25 mM potassium acetate, 100 mM Bicine (pH8.5), 2M citrate] for 12 hours, and the denaturing SDS gels incubated inthe same buffer for 48 hours. At the end of the incubation gels werestained with iodine solution (2% KI and 0.2% 12 in 0.01 N HCl) to detectthe band(s) having starch synthase activity (FIGS. 10 a and 10 b). Thefigures clearly show GBSS activity in all of the waxy-E starches, thatthe activity of GBSS in waxy-E starches is lower than with normal, andthat there is no activity in the lab-isolated waxy starch EX56wx or thecommercial waxy starches.

Western Blotting

An SDS gel (stacker 15 mA and 20 mA for gel) was soaked in 100 mL ofTowbins buffer (25 mM pH 8.3 Tris-acetate; 192 mM glycine) for 10minutes with a nitrocellulose membrane. At the same time, a Towbinstransfer buffer composed of 80 mL of Towbins buffer and 200 mL ofmethanol was made. The soaked gel and nitrocellulose membrane weresandwiched together in a gel holder cassette (composed of a sponge,filter paper, gel, nitrocellulose, and filter paper). Air bubbles wereremoved from the cassette by rolling a glass pipette over the sponge,the gel holder cassette was snapped shut and placed in the transblotmodule. The transfer in Towbins transfer buffer was conducted at 300 mAfor one hour. The nitrocellulose membrane was stained with Ponceuau-S(Sigma, catalog number P7767) 5:45 mis dilution from stock) for 10minutes. The membrane was then incubated in 5% skim milk in TBSbuffer+TWEEN 20 (TBST: 10 mM pH 7.5 Tris; 150 mM sodium chloride; 0.1%TWEEN 20) for 1.5 to 2 hours at room temperature or at 4° C. overnight.After this, the membrane was incubated with primary antibody (1:3000-60kDa) for two hours at room temperature or 4° C. overnight. The membranewas then washed three times with TBST for 15 minutes each time at roomtemperature followed by incubation with secondary antibody (1:3000 goatanti-rabbit IGg with AP conjugate, Biorad, catalog number 1706518) forone hour at room temperature. The membrane was then washed three timeswith TBST for 15 minutes each time at room temperature and the crossreactivity of antibody with GBSS enzyme was detected by developing themembrane using a mixture of 33 μl of a solution of 10 mg of5-bromo-4-chloro-3-indolyl phosphate (BCIP: Sigma, catalog number B6777)in 2 mL of dimethyl formamide and 330 μl of a solution of 200 mg ofnitro blue tetrazolium (NBT) in 2 mL of 70% dimethyl formamide) in 10 mlof alkaline phosphate buffer (100 mM pH 9.5 Tris-sodium hydroxide, 100mM sodium chloride, 5 mM magnesium chloride). The membrane was thenwashed with distilled-deionized H₂O. The reaction was stopped using 5 mMethylene-diamine tetraacetic acid. For detection of the levels of SSIprotein in starch samples, the same procedure as described above wasfollowed using a 1:3000 dilution of antibody for 77 kDa protein.Developed membranes are presented in FIGS. 11 a and 11 b. The membranesclearly show that GBSS protein is present in all of the waxy-E starchesand is absent from the lab isolated waxy starch. Both commercial waxystarches show a very low level of GBSS protein which is likely to befrom contaminating normal starch (Example 1 and FIG. 1) due tocommercial starch isolation practices.

Coomassie Staining of Proteins

At the end of the gel electrophoresis, proteins were stained withcoomassie blue in a buffer containing 43% de-ionized water, 40%methanol, 17% glacial acetic acid, and 0.1% coomassie brilliant blueR-250 (Biorad) for at least 40 minutes. Gels were briefly rinsed withde-ionized water and were de-stained for 20 minutes in a buffercontaining 50% de-ionized water, 40% methanol, and 10% glacial aceticacid (De-stain-I), followed by de-staining for at least 40 minutes in abuffer solution containing 88% de-ionized water, 5% methanol, and 7%glacial acetic acid (De-stain II). At the end of the de-stainingprocedure, the gels were soaked in de-ionized water in order to get ridof any trace amounts of acetic acid trapped in the gel matrix. The wx-E1starches had a level of GBSS during development which appearedsubstantially equivalent to the level of GBSS in the wild type plants,as observed by the level of staining of the bands associated with GBSSin the gel (FIG. 12). The wx-E2 starch had a level of GBSS which wassubstantially reduced as observed by the level of staining of the bandassociated with GBSS in the gel.

Example 13 Identification of a Point Mutation in the Nucleic AcidSequence Encoding Granule-Bound Starch Synthase

The waxy genes from EX385 wild type and EX385wx-E1 mutant seed weresequenced and compared to identify an EMS-induced point mutation. To dothis, total RNA was isolated from immature kernels harvested 17-18 daysafter pollination using standard protocols. The RNA was used as atemplate to synthesize complementary DNA (cDNA) using the enzyme reversetranscriptase by standard protocols. The cDNA was then used as atemplate for the polymerase chain reaction (PCR) to amplify the GBSScoding sequence using two pairs of oligonucleotide primers byconventional methods. The PCR amplified product was then used as atemplate in dideoxynucleotide sequencing reactions that utilizedsequencing primers specific to the GBSS nucleic acid sequence.Techniques for the above methods are described in Current Protocols inMolecular Biology, John Wiley & Sons, Inc. The sequences from EX385 andEX385wx-E1 were compared to each other, as well as to the GBSS sequence(accession number X03935; SEQ ID NO:5) available in Genbank, a publicdatabase. The GBSS sequence from EX385wx-E1 (SEQ ID NO:2) had a singlebase pair change relative to that from EX385 wild type (SEQ ID NO:1),located at position +1643 from the transcription start site. Thismutation changes amino acid 484 from a glycine in EX385 (SEQ ID NO:3) toa serine in EX385wx-E1 (SEQ ID NO:4).

Example 14 Starch Application—Lemon Pie Filling

This experiment was conducted to illustrate the benefits of waxy-Estarch in a lemon pie filling application. Lemon pie fillings wereprepared with normal starches (EX68, Cerestar-USA commercial normalstarch C*Gel 03420), waxy starches (EX68wx, Cerestar-USA commercial waxystarch C*Gel 02430), and waxy-E starches (EX385wx-E1, EX56wx-E1,EX78wx-E1, and EX12wx-E2). The following formulation for lemon piefilling was utilized (Table 17): TABLE 17 Lemon Pie Filling FormulationMass (40 g Ingredient Mass (%) scale) Dry Ingredients Granulated Sugar25.54 10.22 Starch (10% moisture 4.55 1.82 basis) Salt 0.19 0.08 LiquidIngredients Water 46.09 18.44 Lemon Juice 11.71 4.68 Egg Yolk 9.66 3.86Shortening hydrogenated vegetable 2.26 0.91 shortening (melted)

All of the dry ingredients except the starch were combined preceedingthe experiment on a large scale (500 g total prepared and termed thelemon pie filling pre-mix). Liquid ingredients (the water, lemon juice,and egg yolk) for each individual analysis were also combined inadvance. The moisture content of each starch was accounted for in theformulation; all formulations utilized an equal mass of starch on a dryweight basis. Fillings (40 g) were processed using aRapid-Visco-Analyzer as a temperature-controlled mixer. To prepare thefillings, the starch and lemon pie filling pre-mix were added to an RVAsample cup and thoroughly mixed using the stirring paddle to be used forthat sample. The prepared mixtures of dry ingredients and liquidingredients (no shortening) had a pH of 3.3; indicating that the starchwas in a highly acidic environment. The liquid ingredients were thenadded to the RVA sample cup containing the dry ingredients and thestirring paddle agitated to thoroughly suspend the sample solids in theliquid medium. The lemon filling was mixed at 960 rpm for 10 seconds andthen mixed at 160 rpm for the remainder of the first step of the cookingprocess. The first step of the cooking process lasted nine minutesduring which the filling was heated using the following program: theingredients were held at 50° C. for 1 minute, heated from 50° C. to 95°C. for 7.5 minutes, then held at 95° C. for 0.5 minute. After the firststep of the cooking process, the melted vegetable shortening was addedto the filling within 15 seconds. The filling was then stirred at 480RPM for 15 seconds to incorporate the shortening with the otheringredients. Stirring at 160 RPM then resumed and continued for theremainder of the cooking process: fillings were heated at 95° C. for anadditional 2 minutes, cooled to 50° C. over 4.5 minutes, then held at50° C. for 3 minutes. The entire cooking process lasted 19 minutes.Immediately after cooking, the finished lemon pie filling was added to a50 mL tube and placed in a 4° C. refrigerator for storage. Tworeplicates of the experiment were conducted, and the analysis order ofthe second replicate was the opposite of the first replicate in anattempt to eliminate any confounding effect of storage time on theresults.

Lemon fillings prepared with normal starches had formed highly rigidgels within 24 hours of storage at 4° C. and syneresed strongly afterseven days of storage at 4° C. Rheological measurements were notconducted on these samples at either time point. Samples of lemonfillings prepared with waxy starches or waxy-E starches were taken forrheological analysis after 24 hours at 4° C. and after seven days at 4°C.; none of the samples syneresed over the course of the week of storageat 4° C. Frequency and strain dependence of lemon pie fillings weretested using a rheometer (RFSIII Fluids Spectrometer, RheometricScientific, Piscataway N.J.). All fillings were measured at 25° C. Aparallel plate geometry was utilized for testing (50 mm; 0.9 to 1.1 mmgap width); loaded samples were permitted to rest between the plates ofthe rheometer for 10 minutes in order for them to come to 25° C. andalso to reduce the effects of loading on the measurements. A thin filmof oil was applied to the exposed surface of the filling between therheometer plates to minimize moisture evaporation during the testingprocess. Frequency dependence of a filling was always examined first,followed by the strain dependence. The frequency dependence of lemon piefillings was tested between 0.1 and 100 radians per second with aoscillatory strain of 1%. Strain dependence of lemon pie fillings wastested between 0.1 and 1000% deformation at a constant testing frequencyof 1 radian per second. The strain and frequency dependence of the lemonpie fillings stored for 24 hours and seven days at 4° C. prepared withwaxy and waxy-E starches is presented in Table 18 and Table 19,respectively. The results of each replicate are shown.

Fillings made with waxy-E starch stored for 24 hours at 4° C. showedlower frequency dependence than did any of the waxy starches (Table 18and 19), with a 2-3 fold increase in G′ between a frequency of 0.1 and100 radians per second compared to fillings made with waxy starcheswhich generally had a 5-10 fold increase over the same frequency range.The lower frequency dependence of fillings made with waxy-E starchesshows that the waxy-E starches contribute more gel-like character to thefillings than do waxy starches. TABLE 18 Rheology of Lemon PieFillings - Storage for 24 Hours at 4° C. Frequency Dependence StrainDependence 0.1 rad/s 100 rad/s 1% 200% 1000% phase phase phase phasephase G′ angle G′ angle G′ angle G′ angle G′ angle Starch Source (Pa)(deg) (Pa) (deg) (Pa) (deg) (Pa) (deg) (Pa) (deg) wx starch EX68wx 3 2738 40 7 31 3 48 2 64 2 29 38 40 6 33 3 48 2 64 C* waxy 8 18 44 35 13 227 31 3 59 12 17 54 36 16 20 7 33 3 56 waxy-E starch EX56wx-E1 48 5 98 1958 7 41 17 5 67 68 5 132 17 78 6 42 24 6 66 EX385wx-E1 95 5 165 15 108 550 27 6 70 94 3 167 15 107 5 50 28 6 70 EX78wx-E1 76 5 142 17 88 6 22 265 70 91 4 162 15 102 5 46 29 7 68 EX12wx-E1 25 11 73 27 33 14 17 24 4 6224 9 74 26 30 12 17 26 4 61

TABLE 19 Rheology of Lemon Pie Fillings - Storage for 7 Days at 4° C.Frequency Dependence Strain Dependence 0.1 rad/s 100 rad/s 1% 200% 1000%phase phase phase phase phase G′ angle G′ angle G′ angle G′ angle G′angle Starch Source (Pa) (deg) (Pa) (deg) (Pa) (deg) (Pa) (deg) (Pa)(deg) wx starch EX68wx 5 22 42 37 9 26 4 43 3 57 7 24 51 36 11 25 5 44 356 C* waxy 16 12 64 30 22 15 11 30 5 53 18 12 65 30 23 15 11 30 5 51waxy-E starch EX56wx-E1 59 5 124 18 69 7 37 23 5 67 73 4 140 17 82 6 4325 7 67 EX385wx-E1 120 4 200 13 133 4 54 33 7 71 113 4 191 14 126 5 5132 6 71 EX78wx-E1 112 4 198 14 126 5 43 38 6 72 118 3 201 13 131 5 45 386 72 EX12wx-E1 44 7 108 22 52 9 24 28 5 64 43 7 104 21 51 9 23 29 6 63

Fillings made with waxy-E starch stored for 24 hours at 4° C. had ahigher elastic modulus at 1% strain than the elastic modulus of fillingsmade with waxy starch, exceeding 4-fold in all cases and roughly 8-10fold for all of the wx-E1 group starches. Additionally, the phase anglesof waxy-E starch fillings at 1% strain were lower compared with thephase angles of fillings made with waxy starch, indicating that a higherproportion of the complex modulus of fillings made with waxy-E starch isattributable to the elastic component of the filling compared to thosefillings made with waxy starch. Thus, the fillings made with waxy-Estarches are considerably different rheologically from those fillingsmade with waxy starch.

For fillings stored for 24 hours at 4° C., the elastic modulus offillings made with waxy-E starches remained higher than the elasticmodulus of fillings made with waxy starch through 1000% strain.Additionally, through 200% strain the fillings made with waxy-E starchmaintained lower phase angles than the fillings made with waxy starch.Thus, fillings made with waxy-E starch not only retained a relatvelyhigh elastic modulus but also a relatively high elasticity (as acomponent of the complex modulus, indicated by the low phase angles)through high deformations compared to fillings made with waxy starch.

Finally, fillings made with waxy-E starches and waxy starches stored 24hours at 4° C. vs. stored for seven days at 4° C. had similar frequencydependent behavior, strain dependent behavior, an elastic modulusmagnitudes, and phase angles. This similarity between the measurementsafter 24 hours and seven days indicates that the filling properties didnot change much over the course of six additional days of storage at 4°C., indicative of the useful low-temperature stability of formulationscontaining waxy-E starch. Large changes in any of these properties wouldhave indicated the development of additional structure in the filling,which would be undesirable for applications requiring extended storageat low temperatures such as pie fillings used for ready-to-eat piesdistributed from a centralized warehouse to retail outlets. Waxystarches themselves have good low temperature stability, but they do notprovide the higher elasticity that waxy-E starches provide.

The waxy-E starches, because of their high elasticity and lowtemperature stability, could additionally act to suspend for fruit orother large particles in food formulations including pies, puddings,soups, yoghurts, sauces, and other foodstuffs. The waxy starch pastes,though viscous, do not form sufficient paste structure to act as auseful suspension aid. Additionally, because of the unique rheologicalcharacteristics of waxy-E starch pastes and gels, they could be used forcoatings and films in foodstuffs such as batter coatings. Once depositedon a surface, a paste of waxy-E starch will have a better tendency thanwaxy starch to cling and adhere to a surface rather than flow withgravity.

Although the examples above contain many specificities, these should notbe construed as limiting the scope of the invention but as merelyproviding illustrations of some of the presently preferred embodimentsof this invention. Various other embodiments and ramifications arepossible within its scope and are readily apparent to those skilled inthe art. Accordingly, the spirit and scope of the invention are to belimited only by the appended claims and not by the foregoingspecification.

1. A maize waxyE starch having a reduced amylose content, wherein: i) said starch has an EM greater than the EM of a waxy starch produced from a waxy maize plant and less than the EM of a starch from a wild type maize plant; ii) said starch has an AP ratio within 0.5 of the AP ratio of starch of said wild type maize plant, and iii) said starch is indistinguishable from starch from said wild type maize plant when stained with iodine.
 2. The starch of claim 1, having an EM at least twice the EM of waxy maize starch.
 3. The starch of claim 1, having an EM of at least 10 Pascals.
 4. The starch of claim 1, wherein the EM is measured after said starch has been cooked as a suspension of starch using a Rapid Visco Analyzer 4 instrument, and instrument conditions specified in the Newport Scientific Method 1 (STD1) Version 5 heating and stirring profile, and stored for 24 hours at 25° C.
 5. The starch of claim 1, characterized by a phase angle below yield strain which is less than the phase angle below yield strain of a waxy maize starch.
 6. The starch of claim 4, having a gel character below yield strain which is: a) greater than the gel character below yield strain of a waxy maize starch; and b) less than the gel character below yield strain of a wild type maize starch.
 7. The starch of claim 1 which, when subjected to a strain below yield strain, has an increase in G′ less than two fold as the oscillatory testing frequency is increased from 0.1 to 100 radians per second.
 8. A maize waxyE starch having a firmness less than 30 g-s and more than 1 g-s after being cooked as a suspension of 10% starch (dry weight %) according to an RVA Standard Method and then stored for seven days at 4° C., wherein said starch has an AP ratio within 0.5 of the AP ratio of a wild type maize starch.
 9. A maize waxyE starch having a resilience of at least 50% after being cooked as a suspension of 10% starch (dry weight %) according to an RVA Standard Method and then stored for seven days at 4° C., wherein said starch has an AP ratio within 0.5 of the AP ratio of wild type maize starch.
 10. A sol or paste comprising the starch of claim
 1. 11. A gel of the starch of claim
 1. 12. A foodstuff comprising the sol or paste of claim 10
 13. A foodstuff comprising the gel of claim
 11. 14. A foodstuff comprising the starch of claim
 1. 15. A method of making a foodstuff comprising admixing edible ingredients with a maize waxyE starch having a reduced amylose content, wherein: i) said starch has an EM greater than the EM of a waxy starch produced from a waxy maize plant and less than the EM of a starch from a wild type maize plant; ii) said starch has an AP ratio within 0.5 of the AP ratio of starch of said wild type maize plant, and iii) said starch is indistinguishable from starch from said wild type maize plant when stained with iodine. 